Process for making enzyme-resistant starch for reduced-calorie flour replacer

ABSTRACT

An enzyme resistant starch type III having a melting point or endothermic peak of at least about 140° C. as determined by differential scanning calorimetry (DSC) is produced in yields of at least about 25% by weight, based upon the weight of the original starch ingredient. A gelatinization stage, nucleation/propagation stage, and preferably a heat-treatment stage are used to produce reduced calorie starch-based compositions which contain the enzyme resistant starch type III. The high melting point of the enzyme resistant starch permits its use in baked good formulations without substantial loss of enzyme resistance upon baking. A gelatinized, starch-based bulking agent having at least 30% by weight of the enzyme-resistant starch may be used in bar-type, extruded, sheeted, or rotary molded food products. The melting enthalpy of the bulking agent may be from about 0.5 to about 4 Joules/g and its water-holding capacity may be less than 3 grams.

RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.10/651,578, filed Aug. 29, 2003, now U.S. Pat. No. 7,531,199, which is aContinuation of U.S. application Ser. No. 10/036,829, filed Jan. 4,2002, now U.S. Pat. No. 6,613,373, which is a continuation of U.S.application Ser. No. 09/413,325, filed Oct. 6, 1999, now U.S. Pat. No.6,352,733, which is a Divisional of U.S. application Ser. No.08/964,224, filed Nov. 4, 1997, now U.S. Pat. No. 6,013,299. The entiredisclosures of prior application Ser. No. 10/651,578, filed Aug. 29,2003, prior application Ser. No. 10/036,829, filed Jan. 4, 2002, priorapplication Ser. No. 09/413,325, filed Oct. 6, 1999, and priorapplication Ser. No. 08/964,224, filed Nov. 4, 1997 are considered to bepart of the disclosure of this application and are hereby incorporatedby reference herein in their entireties.

FIELD OF THE INVENTION

This invention relates to the production of enzyme-resistant starch inhigh yield for a reduced-calorie flour replacer. Doughs and cookiescontaining the enzyme-resistant starch are also contemplated by thepresent invention. This invention also relates to reduced-calorie bakedgoods which contain the enzyme-resistant starch for substantial caloriereduction.

BACKGROUND OF THE INVENTION

Enzyme-resistant starch (RS) is a fraction of starch not digested in thesmall intestine of healthy individuals. Microflora may partially fermentcertain types of resistant starch in the large bowel. According to adoctoral thesis by Relinde Eerlingen entitled “Formation, Structure andProperties of Enzyme Resistant Starch,” Katholieke Universiteit teLeuven (February 1994), enzyme-resistant starch may be defined as thesum of starch and products of starch degradation not absorbed in thesmall intestine, and it may be classified into four types. Physicallyinaccessible starch, which is locked in the plant cell, is classified astype I resistant starch. It is a fraction which can be found infoodstuffs with partially milled grains and seeds and legumes. Nativegranular starch found in uncooked ready-to-eat starch-containing foods,such as in bananas, is classified as type II resistant starch. Enzymesusceptibility of type II resistant starch is reduced by the highdensity and the partial crystallinity of the granular starch. The amountof type I and type II resistant starches is generally less than about12% by weight; based upon the total amount of uncooked or raw starchcontained in the starch source. However, the type I and type IIresistant starches have low melting points, do not survive a bakingprocess, and do not exhibit good baking functionality. For example,granular starches in the presence of excess water melt at a temperatureof about 80° C. to about 100° C., which is generally below bakingtemperatures for cookies and crackers. Additionally, yields of resistantstarch substantially greater than 12% by weight of the original starchcomponent are desirable for the mass production of baked products havingsubstantially reduced calorie content.

Starch may be treated to obtain an indigestible starch fraction.Depending upon the type of treatment, a type III resistant starch or atype IV resistant starch may be produced. An indigestible starchfraction which forms after certain heat-moisture treatments of thestarch, which may be present in, for example, cooled, cooked potatoesand canned peas or beans, is type III enzyme-resistant starch.

In type IV resistant starch, the enzyme resistance is introduced bychemically modifying or thermally modifying the starch. The modificationmay be the formation of glycosidic bonds, other than alpha-(1-4) oralpha-(1-6) bonds, by heat treatment. Formation of these otherglycosidic bonds may reduce the availability of starch for amyloliticenzymes. These types of bonds may be present, for example, in productsof caramelization and products of Maillard reactions.

U.S. Pat. No. 5,330,779 to Watanabe discloses a food additive which isslowly absorbed and digested, comprising a mixture of starchescomprising a starchy material having a high amylose content and amodifier which modifies the enzymatic reaction ratio with amylase, suchthat it is not more than 95% digested, as compared to an unmodifiedstarch mixture. The modifier may be a saccharide or a fatty acidcompound.

U.S. Pat. Nos. 5,364,652 and 5,472,732 and European patent applicationpublication 443,788A1 (published Aug. 28, 1991), each to Ohkuma et al.,disclose the production of indigestible dextrins or pyrodextrins byheat-treating potato starch in the presence of an acid and then refiningthe product. According to U.S. Pat. Nos. 5,364,652 and 5,472,732,attempts to increase the amount of pyrodextrin produced by increasingthe reaction time and reaction temperature result in a coloredsubstance, release a stimulative odor, and result in a product which isnot practically useful. Each of the U.S. patents and the European patentpublication disclose refining of the pyrodextrin by the use ofhydrolysis with alpha-amylase, followed by separation of the dextrinfraction from the digestible components by continuous chromatographywith use of an ion-exchange resin.

In addition, the digestibility of starch may be reduced by cross-linkingor the presence of various substituents such as hydroxypropyl groups.However, the chemical or thermal modification of the starch, whichresults in a type IV resistant starch, often affects the bakingcharacteristics of the starch. In addition, chemically or thermallymodified starches may exhibit undesirable flavors or colors when used insubstantial amounts in baked goods. Legal limitations by the U.S. Foodand Drug Administration (FDA) have also been placed upon the use ofvarious chemically modified starches in baked goods.

However, to produce enzyme-resistant starch type III, heat-moisturetreatments of the starch create crystalline regions, without theformation of glycosidic bonds other than alpha-(1-4) or alpha-(1-6)bonds. The type III resistant starch is thermally very stable, which ishighly advantageous for producing reduced-calorie baked goods. If thecrystal structure which provides enzyme resistance is destroyed or meltsduring baking, and if the crystal recrystallizes into a lower-meltingform which is not enzyme resistant, then calorie reduction will not beachieved in the baked product. According to the Eerlingen dissertation,when RS type III is heated in the presence of water, an endotherm isrevealed at about 150° C., with enthalpy values ranging from 8 mJ/mg to30 mJ/mg. Heating to 180° C., it is reported, leads to partial thermaldegradation of the RS chains. During cooling, an exotherm with anenthalpy value of about −22 mJ/mg, starting at about 60° C., can beobserved. The exotherm has been attributed to reassociation of theresistant-starch chains.

Reported chain lengths for resistant starch type III vary between anaverage degree of polymerization, DP_(n), of 22 and 65 glucose residues,with the chains being linear. Accordingly, RS type III is reported asconsisting of short linear segments of alpha-glucans arranged in acrystalline structure.

To produce enzyme-resistant starch type III from native starch granules,the starch has to be gelatinized and then retrograded. Factors whichaffect the yield of enzyme-resistant starch type III include: amylosecontent of the starch, the number of autoclaving-cooling cycles used toform the RS type III, the water content of the starch, the autoclavingtemperature, and the presence of complexing lipids. It has been reportedthat higher amylose-content starches result in increasedresistant-starch yield. According to Eerlingen, high yields of more than20% resistant starch can be obtained from autoclaved amylomaize starchcontaining 70% amylose. This yield, it is stated, can even be raised tolevels of 40% by increasing the number of autoclaving-cooling cycles upto 20 cycles. A starch:water ratio of 1:3.5 is disclosed as providing anoptimum in resistant-starch yield. The effect of autoclaving temperatureupon resistant-starch yield has been reported to depend upon the starchtype. According to Eerlingen, increasing the autoclaving temperaturefrom 100° C. to 134° C. increased the RS yield for wheat starch, but didnot significantly affect the yield for amylomaize starch. It is alsodisclosed that the formation of amylose-lipid complexes, due to theaddition of an excess of complexing lipids, decreases resistant-starchyields.

Several methods are available for the in vitro determination ofresistant starch. The resistant-starch levels and yields determined invitro depend upon the method used. The methods differ in the enzymesused and the temperature-time conditions of incubation. Lowerresistant-starch yields are obtained when more severe conditions areapplied, such as higher incubation temperatures, longer incubationtimes, and higher enzyme levels. For example, in one procedure, starchis incubated for 16 hours with pancreatin at 37° C. In anotherprocedure, known as the Prosky method, a fiber fraction is isolated inthe starch samples after incubation with different enzymes, such as aheat-stable alpha-amylase at 100° C. In this residue, RS was determinedas the starch available for amyloglucosidase digestion at 60° C., onlyafter solubilization with 2N potassium hydroxide. The resistant-starchyields in the more severe or Prosky method are lower than when the firstmethod is used. When using incubation temperatures of 100° C., thestarch is gelatinized and RS type II is not quantified. Additionally,retrograded amylopectin, which exhibits a melting temperature of about50° C., and amylose-lipid complexes, with melting temperatures in therange of 90-110° C., are easily hydrolyzed when incubated with aheat-stable alpha-amylase at 100° C. However, hydrolysis with pancreatinat 37° C., according to the first method, depends upon incubation time,enzyme:substrate ratio, and on the degree of organization of thesubstrate. Additionally, retrograded amylopectin and amylose-lipidcomplexes, which melt above 37° C. but below 100° C., may falsely beincluded as a higher-melting (e.g. 150° C.) RS type III.

Thus, although Eerlingen discloses that yields of more than 20% and upto 40% of resistant starch have been obtained, these yields areapparently determined by the much less stringent method of usingpancreatin at 37° C. The yields would include production of thehigh-melting (150° C.) RS type III as well as lower-melting retrogradedamylopectin, amylose-lipid complexes, and any other starch complexeswhich melt above 37° C. The substantial difference in yields obtainedusing the two different in vitro resistant-starch determination methodsis demonstrated in the Eerlingen thesis at pages 107-108. Measurement ofresistant-starch contents was made using pancreatin and amyloglucosidaseat 37° C. This method resulted in the measurement of the total RScontent which included three types of resistant starch: physicallyinaccessible starch, resistant-starch granules, and retrograded starch.The total RS content (for the three types of resistant starch) washighest for high-amylose corn starch and is reported as 83.2% of drymatter. However, when the more stringent conditions used to determinedietary fiber contents (DF contents) were used (Termamyl, a thermostablealpha-amylase from Bacillus Licheniformis, at 100° C. andamyloglucosidase at 600° C.), the dietary fiber content was only 17% ofdry matter. It is further concluded that the dietary fiber (DF) of thehigh-amylose corn starch probably consisted of very resistant starchgranules or granule remnants rather than retrograded starch or resistantstarch type III.

Additionally, for a sample of extruded retrograded high-amylose cornstarch (ERHA), the RS content was 29.5% of dry matter, but the DFcontent was only 15.5% of dry matter. It was concluded that the DF ofERHA most likely consisted of retrograded amylose, because retrogradedamylopectin melts at 40 to 60° C. Eerlingen further reports that theERHA sample had a drastic impact on moisture binding, required muchlonger dough-mixing times, and gave significantly smaller loaf volumes,compared to control breads made with wheat flour.

According to Eerlingen, resistant-starch yields depend upon storagetemperature, between the glass transition and melting temperature, andon storage time to a great extent. Nucleation, it is disclosed, isfavored at temperatures far below the melting temperature of the amylosecrystals but above the glass transition temperature, while propagationis limited under these conditions. However, at temperatures far abovethe glass transition temperature but below the melting temperature, itis disclosed, propagation is favored, while nucleation is limited.

In an attempt to obtain a maximum yield of resistant starch in a minimumof time, Eerlingen conducted nucleation at 0° C., followed bypropagation at a higher temperature of 68° C. or 100° C. The greatestyields were expected for incubation at 0° C. (30 minutes), wherenucleation is favored, and subsequent storage at 100° C., wherepropagation is favored. However, it was observed that the yield ofresistant starch formed at 100° C. after incubation at 0° C. (30minutes) did not increase significantly. It is further reported thatyields did not increase even after incubation times at 100° C.Resistant-starch yield did not significantly increase, when incubationof autoclaved water-starch mixtures was conducted at 68° C., afterstorage at 0° C.

According to Eerlingen, the results demonstrate that to achieve a highamount of RS in a relatively short time, a two-step procedure withsubsequent incubation at 0° C. and a higher temperature is not the bestway to proceed. A higher amount of resistant starch (about 10% RS forwheat starch) can be obtained by a single-step procedure at 100° C., butstorage times of three days or more are necessary (see pages 62-8 of theEerlingen dissertation). The enzyme-resistant starch contents weredetermined using the heat-stable-alpha-amylase at 100° C. and anamyloglucosidase at 60° C. Differential scanning calorimetry of theisolated RS residues showed a melting endotherm with a peak temperatureat about 155° C.

U.S. Pat. No. 5,051,271 and corresponding International patentpublication no. WO 91/07106 (published May 30, 1991), each to Iyengar etal., disclose the production of a retrograded starch product for use asa bulling agent, extender, or substitute for sugar, flour, or fat infoods. A starch sample is dispersed in an aqueous medium containing atleast 80% by volume of water, to obtain a suspension having up to about10% (w/v) of starch. The dispersion is then incubated at an elevatedtemperature of preferably about 60°-120° C. for a period of timesufficient to cause retrogradation to occur, for example, about 5 toabout 10 hours. The product is then cooled and incubated at a lowertemperature of about 4° to about 20° C. for about 0.5 to about 4 days.According to Iyengar et al., at this point at least 50% by weight of thestarch consists of crystalline regions. The first step of the process,it is disclosed, can be accelerated by enzymatic conversion ofamylopectin to amylose prior to retrogradation, because retrogradationof amylose is retarded by the presence of the amylopectin in the starch.Digestibility of the product is determined using the less stringentmethod which employs pancreatin with incubation at 37° C. Foods whichcan be formulated using the retrograded starch products, it isdisclosed, include cookies, fudge, brownies, low-fat margarine spreads,and frozen desserts. The water-holding capacity of amylose, it isdisclosed, was found to be 6.4 g/g. However, retrogradation andenzymatic treatment resulted in a decreased level of water-holdingcapacity. The water-holding capacity for retrograded amylose (RA) wasfound to be 3.4 g/g, and was 2.0 g/g for crystalline water-insolubleenzyme-modified retrograded amylose (EMRA). The melting temperature ofthe retrograded amylose, as determined by differential scanningcalorimetry (DSC), is not disclosed. However, cooling and incubation at4° C. to 20° C. would promote the crystallization of amylopectin.

International patent publication no. WO 90/15147 (published Dec. 13,1990) to Pomeranz et al. discloses the production of purifiedresistant-starch products having at least 50% resistant-starch contentby forming a water-starch suspension wherein the ratio of starch towater is approximately 1:2 to 1:20 and heating the water-starchsuspension in an autoclave at temperatures above 100° C. to ensure fullstarch gelatinization. The mixture is then cooled to allow amyloseretrogradation to take place. As indicated in Example 1, autoclaving wasat either 121° C., 134° C., or 148° C. Each of the samples was allowedto cool to room temperature overnight for the retrogradation to takeplace. It is reported that best results were obtained at a temperatureof 134° C., with four heating and cooling cycles and a starch:waterratio of 1:3.5. The resistant starch is purified by comminuting thestarch gel and mixing it with an amylase to digest non-resistant starchfractions, leaving resistant starch. The amylase is inactivated by heattreatment above 100° C. Resistant-starch yield from amylomaize VII,using Termanyl or Takalite bacterial alpha-amylase, is reported as16.2%, based upon the weight of the starch used to prepare the sample inExample 3, wherein one cooling cycle to room temperature overnight wasutilized. It is also reported in Example 13 that cookies prepared usingstandard cookie flour supplemented to provide 3%, 5%, and 7%concentrations of 70% purified (i.e. after removal of 30% by weight ofamylase digestible starch) resistant starch showed reduced cookiediameter and paler cookie color, compared to cookies prepared withsimilar levels of wheat bran or soy fiber. Both the crude, heat- andmoisture-treated starch and purified resistant-starch products arereported as having a transition temperature T_(p) (the temperature atthe maximum of the endothermic melting peak) of from 149.1° C. to 154.5°C.

U.S. Pat. No. 5,281,276 to Chiu et al. discloses the preparation of astarch product containing amylase-resistant starch, by gelatinizing aslurry of a starch that contains amylose in an amount greater than 40%,treating the gelatinized starch with a debranching enzyme to effectessentially complete debranching, deactivating the enzyme, and isolatingthe starch product by drying, extrusion, or crystallization by theaddition of salt. According to Chiu et al., the method does not requirerepeated cycles of gelatinization and incubation at low temperatures toproduce the resistant-starch product. The starch product, it isdisclosed, contains a minimum of about 15% resistant starch. The dietaryfiber is determined by using the Prosky method, wherein incubation withTermanyl is conducted at 100° C. To isolate the starch product, aninorganic salt is added to the starch dispersion, and the mixture isincubated at 50° C. to 100° C. The salt acts to help draw out the waterof gelatinization, it is disclosed, thereby permitting the associationof the linear starch molecules and the formation of amylase-resistantstarch. However, the process requires large amounts of salt, which mayadversely affect taste. The salts are added to the deactivated starchslurry in a minimum of 10% of the solids content.

U.S. Pat. Nos. 5,374,442, 5,387,426, and 5,395,640, each to Harris etal., disclose the preparation of a fragmented starch precipitate for usein preparing reduced-fat foods. In the process of U.S. Pat. No.5,395,640, a debranched amylopectin starch is precipitated and thenfragmented. The debranched amylopectin starch may be derived from astarch which contains amylopectin, for example, common corn starch andwaxy maize starch, by gelatinizing the starch, followed by treatmentwith a debranching enzyme, such as isoamylase or pullulanase, andprecipitation of the debranched starch. To form the precipitate, thesolution is cooled, for example, to ambient temperature, to reduce thesolubility of the debranched starch. The precipitate may then be heatedto about 70° C., while in contact with a liquid medium, to dissolve atleast a portion of the precipitate. Reprecipitation by cooling of thesuspension/solution may then be employed. Repetition of the dissolvingand the reprecipitation, it is disclosed, tends to improve thetemperature stability of the resulting aqueous dispersion. In Example15, a water bath was used to heat debranched waxy maize starch to 99°C., the temperature was held there for 60 minutes, then the starch wascooled to 4° C. and held at that temperature for 60 minutes. The cycleof heating and cooling was repeated a total of eight times. DSC analysisindicated a melting-onset temperature of 46° C. to 47° C. and amelting-end temperature of 121° C. to 132° C., depending upon the numberof crystallization cycles completed. It was also observed that a majorpeak centered at about 115° C. increased, while the size of the peak atabout 85° C. was reduced, as the number of crystallization cyclesincreased.

In the process of U.S. Pat. No. 5,374,442, a starch having both amyloseand amylopectin is gelatinized to allow preparation of pure amylose as apermeate of membrane filtration. The amylose is precipitated,recrystallized, and then fragmented to form an aqueous dispersion foruse in replacing fat. The solution of amylose is allowed to form aprecipitate by cooling to ambient temperature to reduce the solubilityof the amylose. Subjecting the precipitate to recrystallization, by slowheating and slow cooling over a temperature range of about 50° C. to100° C., is disclosed as making the precipitate much more stable (i.e.,resistant to solubilization) at elevated temperatures.

In the process of U.S. Pat. No. 5,387,426, retrograded, hydrolyzed,heat-treated, and fragmented amylose starch is made by the sequentialsteps of gelatinization, retrogradation, hydrolysis, heat treatment, andfragmentation of a starch material containing amylose. The solution ofgelatinized, optionally debranched starch is allowed to form aprecipitate of retrograded starch by cooling from the temperature atwhich the starch is pasted, to reduce the solubility of the gelatinizedstarch. The solution, it is disclosed, is typically held at an elevatedtemperature, for example, 65° C. to 90° C., until substantialequilibrium is achieved between the supernatant and the precipitate.Heating of the particles (for example, to about 70° C.), it isdisclosed, and then cooling of the suspension/solution tends to make theparticles resistant to melting or dissolving, when an aqueous dispersionof the particles is exposed to heat in processing. In Example 1 of U.S.Pat. No. 5,387,426, a high-amylose starch was solubilized in water atabout 150° C. The resulting solution was cooled to room temperature(about 25° C.) and allowed to stir for 20 hours, during which time athick mass of crystals precipitated. The crystals were hydrolyzed inacid, and then insoluble product was isolated by centrifugation. It isreported that the DSC endotherm of the hydrolysis product was verybroad, beginning at about 80° C. and ending at about 138° C. Twodomains, peaking at about 100° C. and about 115° C., respectively, werereported. The material in the higher temperature domain, it isdisclosed, could be isolated by washing the material with water at atemperature above 100° C., for example, from about 105° C. to about 110°C. In Example 2 of U.S. Pat. No. 5,387,426, a product having a DSCendotherm having a single domain which peaked at about 120° C. isreported.

Thus, even though the methods of Harris et al.'s U.S. Pat. Nos.5,374,442, 5,387,426, and 5,395,640 involve subjecting starches togelatinization, precipitation, and optionally heating and coolingcycles, the resulting retrograded amylose products are reported in thelatter two patents to have DSC endothermic peaks at no more than about120° C. The results obtained in the Harris et al. patents indicate thatretrograded amylose may have melting points above 100° C. but below theapproximately 150° C. melting point of RS type III. Accordingly,reported resistant-starch content or dietary fiber content, determinedeven by the more stringent Prosky method wherein treatment at 100° C. isutilized, may often include crystalline forms which melt substantiallybelow the approximately 150° C. endothermic peak of RS type III.

Heat-treating of dehydrated starch for a time and at a temperature toinhibit the starch or flour is disclosed in PCT International PatentPublication Nos. WO 96/22073 and 96/22110 (each published Jul. 25,1996). The thermally inhibited starches of WO 96/22073 are used incosmetic compositions as emulsifiers, thickeners, and aesthetic controlagents. The heat treatment, it is disclosed, improves the starch'sviscosity stability when dispersed in water. The thermally inhibitedstarches of WO 96/22110 are used in pharmaceutical products as adiluent, filler, thickener, and the like.

Repeated heat-moisture treatment is reported as being associated with adecrease in the hydrolysis limit of pancreatic alpha-amylase andincreased formation of resistant starch in Kobayashi, T.,“Susceptibility of heat-moisture-treated starches to pancreaticalpha-amylase, and the formation of resistant starch by heat-moisturetreatment,” Denpun-Kagaku, 40 (3) pp. 285-290 (1993). However, thestarch is not a heat-stable, indigestible starch.

Production of resistant starch in legumes by steam-cooking anddry-heating is disclosed in Tovar et al., “Steam-Cooking and Dry HeatingProduce Resistant Starch in Legumes,” J. Agric. Food Chem. 44, pp2642-2645 (1996). Seeds were steam-cooked under pressure by placing themin an open glass flask which was then autoclaved at 121° C. for 15minutes. This pressure treatment was also carried out in capped flasks,in order to prevent direct steam/seed contact (“dry pressure heating”).According to Tovar et al., isolates from steam-heated legumes were richin indigestible (resistant) starch (19-31%, dmb), a fact not observedwhen raw seeds were used. Retrogradation, it is disclosed, is suggestedas being the major mechanism behind the reduction in digestibility.Prolonged steaming, as well as short dry pressure heating, decreased theenzymatically assessed total starch content of whole beans by 2-3%(dmb), indicating that these treatments may induce formation of othertypes of indigestible starch.

The present invention provides a process for producing a starch-basedcomposition comprising an enzyme-resistant starch type III which has amelting point of at least about 140° C., as determined by differentialscanning calorimetry (DSC). The very high melting-point,enzyme-resistant starch may be produced on a batch, semi-continuous orcontinuous basis in high yields of at least about 25% by weight, basedupon the weight of the original starch ingredient, as determined by thestringent Prosky method. The enzyme-resistant starch is produced underconditions to avoid discoloration, malodors, and substantial productionof lower-melting amylopectin crystals, lower-melting amylose crystals,and lower-melting amylose-lipid complexes. The starch-based compositionscomprising the high-melting RS type III of the present invention exhibitunexpectedly superior baking characteristics, such as enhanced cookiespread, golden brown color, pleasant aroma, and surface cracking, whichare comparable to those achieved with conventional wheat flour. Thewater-holding capacity of the starch-based composition is comparable tothat of conventional wheat flour. The high melting point of theenzyme-resistant starch, as measured by DSC, permits its use in bakedgood formulations without substantial loss of enzyme resistance uponbaking. It may therefore be used for the production of reduced-caloriebaked goods such as cookies.

The present invention also provides a method for heat-treating anenzyme-resistant starch composition. The enzyme-resistant starchcomposition which is subjected to the heat-treatment may compriseenzyme-resistant starch type I, II, III, or IV. The heat-treatmentsubstantially increases the yield of enzyme-resistant starch or dietaryfiber content of the composition and enhances its bakingcharacteristics.

SUMMARY OF THE INVENTION

The present invention provides a process for producing enzyme-resistantstarch type III having an endothermic melting peak of at least 140° C.,preferably at least 145° C., most preferably at least about 150° C., asdetermined by modulated differential scanning calorimetry (MDSC). Thevery-high-melting, enzyme-resistant starch component is substantiallyunaltered by baking, that is, it remains substantially enzyme resistantand exhibits a reduced caloric value of less than about 0.5Kcalories/gram (100% by weight RS type III, having a melting point orendothermic peak temperature of at least 140° C.), as determined byfiber analysis after baking.

Enthalpy values for the isolated high-melting enzyme-resistant starchmay range from greater than about 5 Joules/g, preferably from about 8Joules/g to about 15 Joules/g, at a temperature of from 130° C. to about160° C. The enthalpy value may depend upon the perfection of thecrystals or the presence of amorphous regions or sections in thecrystal. Higher degrees of perfection and higher enthalpy values may beachieved by increasing the number of cooling and heating cycles duringproduction of the crystals. The enzyme-resistant starch may havecrystalline chains of at least about 20 glucose units, preferably atleast about 100 glucose units, derived from amylose or amylopectin.

The starch may be any starch having both amylose and amylopectin, buthigh-amylose starches such as amylomaize starch, or legume starch, suchas wrinkle pea starch, are preferred. In embodiments of the invention,brewer's spent grain may be used as a low-cost source of amylose for theproduction of the resistant starch type III.

The very-high-melting, enzyme-resistant starch is produced in highyield, as determined by the more stringent Prosky method for thedetermination of dietary fiber. High yields of the enzyme-resistantstarch may be achieved on a continuous, consistent basis usingrelatively short crystal-nucleation and crystal-propagation times. Highyields of the enzyme-resistant starch are achieved using processingconditions which avoid substantial discoloration or the production ofcomponents which impart offensive odors to the product. In addition, theproduction of lower-melting-point amylopectin crystals,lower-melting-point amylose crystals, and lower-melting-pointamylose-lipid complexes, all of which tend to reduce yield of thehigh-melting resistant starch type III crystals, is substantiallyavoided in the process of the present invention.

In accordance with the method of the present invention, in a first stageof the process, a starch ingredient is heated in the presence of waterto at least substantially, preferably completely, gelatinize the starch.The gelatinization is conducted at a temperature above the melting pointof any amylose-lipid complex which may be present in the starchingredient, but below the melting point of the enzyme-resistant starch.Type III. In preferred embodiments, the starch is pasted as well asgelatinized. Exemplary starch-gelatinization temperatures which may beemployed may range from about 110° C. to about 130° C. The weight ratioof starch to water may range from about 0.15:1 to about 1:1, preferablyfrom about 0.4:1 to about 0.7:1, during gelatinization as well as duringthe subsequent nucleation and propagation steps.

A second stage of the process involves at least one cycle of crystalnucleation and propagation. In a critical cooling step, the gelatinizedstarch is cooled to a crystal nucleating temperature above the meltingpoint of amylopectin starch. The nucleating temperature employed is alsopreferably not favorable to nucleation of any amylose-lipid complexwhich may have been present in the starch ingredient. By not coolingbelow the melting point of amylopectin, nucleation and growth ofamylopectin crystals, which are believed to compete with or impede thenucleation and growth of high-melting amylose crystals, is avoided.Exemplary nucleating temperatures range from about 55° C. to about 100°C., preferably from about 60° C. to about 80° C. The gelatinized starchis maintained at the nucleating temperature for a period of timesufficient to nucleate a substantial amount of crystals of thehigh-melting point, enzyme-resistant starch. Exemplary nucleation timesrange from about 0.5 hours to about 3 hours. Longer nucleation times,for example up to about 24 hours, may be used but do not substantiallyincrease yields. The rate of cooling of the gelatinized starch to thenucleating temperature should be as fast as possible and may be at leastabout 1° C./min, preferably at least about 3° C./min, most preferably atleast about 4° C./min.

After maintaining the gelatinized starch at the nucleating temperature,the temperature of the gelatinized starch is raised above the meltingpoint of any amylose-lipid complexes, to a crystal-propagatingtemperature which is below the melting point of the desiredenzyme-resistant starch. Thus, any amylose-lipid complex which may havebeen formed during nucleation would be remelted during propagation orgrowth of the enzyme-resistant starch crystals. The temperature may beraised from the nucleating temperature to the crystal-propagatingtemperature at a rate of from at least about 1° C./min, preferably atleast about 3° C./min, most preferably at least about 4° C./min to avoidany substantial propagation of undesirable crystals, such asamylose-lipid complexes. Exemplary crystal-propagating temperatures forgrowing crystals of the enzyme-resistant starch may range from about115° C. to about 135° C., preferably from about 120° C. to about 130° C.Exemplary times for maintaining the temperature at thecrystal-propagating temperature are generally less than about 12 hours,preferably less than about 5 hours, most preferably from about 0.5 toabout 3 hours.

The steps of cooling the gelatinized starch, maintaining the gelatinizedstarch at the nucleating temperature, raising the temperature of thegelatinized starch to a crystal-propagating temperature, and maintainingthe temperature at the crystal-propagating temperature to grow crystalsmay be sequentially performed in at least one cycle, preferably from twoto four cycles, to increase yields of the high-melting enzyme-resistantstarch. In embodiments of the invention, up to about 10 to 12 cycles maybe utilized.

After the last step of crystal propagation, the gelatinized starch maybe cooled to about room temperature and then dried. The temperaturecycling increases yield and achieves high calorie reduction, without theneed to isolate the high-melting enzyme-resistant starch type III. Thedried composition may be used as a bulking agent, or flour substitute orreplacer, thereby avoiding crystal solids losses and increased costsassociated with isolation of the high-melting resistant starch type III.

In embodiments of the invention, a debranching enzyme such aspullulanase may be used to increase the yield of the high-meltingenzyme-resistant starch type III. The debranching may occur prior to, orpreferably after, a substantial amount of high-melting enzyme-resistantstarch type III has been propagated.

In other embodiments of the invention, seed crystals of the high-meltingenzyme-resistant starch type III may be admixed with the gelatinizedstarch above the melting point of amylopectin crystals and above themelting point of any amylose-lipid complexes, but below the meltingpoint of the high-melting enzyme-resistant starch, to nucleate crystalsof the enzyme-resistant starch type III.

In preferred embodiments, a third stage may be conducted, involving heattreatment of the enzyme-resistant starch type III product obtained fromthe second-stage nucleation/propagation temperature cycling. The heattreatment substantially increases the amount or yield ofenzyme-resistant starch or total dietary fiber. The heat treatment alsosubstantially improves the baking characteristics or bakingfunctionality of the second-stage product. The higher enzyme-resistantstarch content or dietary fiber content is achieved, withoutsubstantially adversely affecting the content of enzyme-resistant starchtype III which melts at a temperature of at least about 140° C. The heattreatment is believed to increase the amorphous or non-crystalline,enzyme-resistant starch content of the second-stage product.

The heat treatment may be conducted at a temperature of from about 100°C. to about 140° C., preferably from about 125° C. to about 135° C.,most preferably from about 128° C. to about 132° C. Heat-treatment timesmay range from about 5 minutes to about 6 hours, preferably from about30 minutes to about 90 minutes, most preferably from about 50 minutes toabout 70 minutes. The moisture content of the enzyme-resistant starchduring heat treatment may be from about 1% by weight to about 30% byweight, preferably from about 14% by weight to about 24% by weight, mostpreferably from about 16% by weight to about 20% by weight.

In other embodiments of the invention, the heat treatment may also beused to treat other enzyme-resistant starch-containing compositions.Thus, compositions comprising enzyme-resistant starch type I, II, or IVmay be subjected to the heat treatment of the present invention toincrease the dietary fiber content or enzyme-resistant starch content ofthe compositions and to improve their baking characteristics.

The products obtained by the process of the present invention maycomprise at least about 25% by weight, preferably at least 30% byweight, most preferably at least about 35% by weight, ofenzyme-resistant starch type III as determined by the rigorous Proskymethod. The balance of the product may comprise gelatinized, amorphous,or non-crystallized starch, a substantial portion of which may be enzymeresistant and contribute to the dietary fiber content of the resultingproduct.

The resulting product may be used as a bulking agent or flour substitutein the production of reduced-calorie baked goods. Even though theproduct contains high amounts of gelatinized starch, it exhibitsexcellent cookie-baking characteristics in terms of oven spread, edgecontour, oil release, surface cracking, odor, color or browning,mouthfeel, and texture. It may be used alone or preferably incombination with non-gelatinized, conventional wheat flour to obtaindoughs for the production of reduced-calorie baked goods such ascookies.

The doughs of the present invention comprise at least about 12.5% byweight of enzyme-resistant starch having a melting point of at leastabout 140° C., said weight percentage being based upon the total starchcontent of the dough. The amount of gelatinized starch-based bulkingagent may generally be at least about 25% by weight, for example atleast about 40% by weight, preferably from about 50% by weight to about75% by weight, based upon the total weight of the gelatinized buildingagent and ungelatinized wheat flour.

In preferred embodiments, the water-holding capacity of the gelatinized,enzyme-resistant bulking agent or flour substitute is comparable to thatof conventional ungelatinized wheat flour, so as to avoid excessive ortoo little cookie spread upon baking. In embodiments of the invention,the water-holding capacity of the resistant-starch ingredient is lessthan 3 grams of water per gram of dry matter. Exemplary water-holdingcapacities are less than about 250% by weight, and preferably range fromabout 100% by weight to about 200% by weight (e.g., 1 gram water/gramdry resistant-starch ingredient to 2 grams water/gram dryresistant-starch ingredient). The enthalpy of the enzyme resistantbulking agent or flour substitute used in baking, may range from about0.5 J/g to about 4 J/g, at a temperature within the range of about 130°C. to about 160° C., generally from about 1 J/g to about 3 J/g, forexample about 2.5 J/g, based upon the weight of the bulking agent orflour substitute.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a modulated differential scanning calorimetry (MDSC) curvefor an enzyme-resistant starch type III ingredient or bulking agent,obtained in Example 1A using a nucleation temperature of about 70° C.and a propagation temperature of about 130° C., in accordance with thepresent invention.

FIG. 1B shows a MDSC curve for an isolated enzyme-resistant starch typeIII, obtained in Example 1B, which was isolated from the ingredient orbuilding agent of Example 1A.

FIGS. 2A and 2B show MDSC curves for two samples (A and B) of anenzyme-resistant starch type III ingredient or bulling agent, obtainedin Example 2A using a nucleation temperature of about 58° C. and apropagation temperature of about 120° C., in accordance with the presentinvention.

FIG. 3 shows a MDSC curve for an enzyme-resistant starch type IIIingredient or bulking agent, obtained in Example 3A, which wassubsequently treated with pullulanase, the bulking agent being preparedusing a nucleation temperature of about 70° C. and a propagationtemperature of about 130° C.

FIG. 4 shows a MDSC curve for a heat-treated enzyme-resistant starchtype III ingredient or bulking agent obtained in Example 4 byheat-treating a sample of the bulking agent of Example 1A.

FIG. 5A shows a MDSC curve for an enzyme-resistant starch type IIIingredient or bulking agent control sample, obtained in Example 6 usinga nucleation temperature of about 70° C. and a propagation temperatureof about 130° C.

FIG. 5B shows a MDSC curve for an isolated enzyme-resistant starch typeIII control sample, obtained in Example 6, which was isolated from theingredient or bulking agent of Example 6.

FIG. 5C shows a MDSC curve for an isolated, heat-treatedenzyme-resistant starch, obtained in Example 6 by heat-treating a sampleof the enzyme-resistant starch type III bulking agent of Example 6 at aheat-treating temperature of 130° C. for one hour and at a moisturecontent of 20% by weight, followed by isolation of the RS from theheat-treated bulking agent.

FIG. 5D shows a MDSC curve for a heat-treated enzyme-resistant starchbulking agent obtained in Example 6 by heat-treating a sample of theenzyme-resistant starch type III bulling agent of Example 6 at aheat-treating temperature of 130° C. for one hour and at a moisturecontent of 14.8% by weight.

FIG. 5E shows a MDSC curve for an isolated, heat-treatedenzyme-resistant starch, obtained in Example 6 by heat-treating a sampleof the enzyme-resistant starch type III bulking agent of Example 6 at aheat-treating temperature of 130° C. for one hour and at a moisturecontent of 14.8% by weight, followed by isolation of the RS from theheat-treated bulling agent.

FIG. 5F shows a MDSC curve for an isolated, heat-treatedenzyme-resistant starch, obtained in Example 6 by heat-treating a sampleof the enzyme-resistant starch type III bulking agent of Example 6 at aheat-treating temperature of 130° C. for one hour and at a moisturecontent of 18.4% by weight, followed by isolation of the RS from theheat-treated bulking agent.

FIG. 6 shows a process flow sheet for the production of enzyme-resistantstarch type III using a heat-treatment stage in accordance with apreferred embodiment of the present invention.

FIG. 7 shows a MDSC curve for an isolated, non-heat-treatedenzyme-resistant granular starch type II (Novelose 240) of Example 8.

FIG. 8 shows a MDSC curve for an isolated, heat-treated enzyme-resistantgranular starch type II, obtained in Example 8 by heat-treating a sampleof the enzyme-resistant granular starch type II (Novelose 240) bulkingagent of Example 8 at a heat-treating temperature of 130° C. for onehour, followed by isolation of the RS from the heat-treated bulkingagent.

FIG. 9 shows a MDSC curve for a non-heat-treated enzyme-resistantretrograded starch (an RS type III ingredient, Novelose 330) bulkingredient of Example 9.

FIG. 10 shows a MDSC curve for a heat-treated enzyme-resistantretrograded starch (an RS type III ingredient) bulk ingredient, obtainedin Example 9 by heat-treating a sample of the enzyme-resistantretrograded starch (Novelose 330) bulking agent of Example 9 at aheat-treating temperature of 130° C. for one hour.

FIG. 11 shows a MDSC curve for a heat-treated enzyme-resistant starchtype III bulking agent of Example 10, Sample 2.

DETAILED DESCRIPTION OF THE INVENTION

High-melting-point, enzyme-resistant starch type III (also referred toas RS III) is obtained in high yield by using a nucleating temperatureabove the melting point of amylopectin crystals. The enzyme-resistantstarch type III has a melting point or endothermic peak temperature ofat least about 140° C., preferably at least about 145° C., mostpreferably at least about 150° C., as determined by modulateddifferential scanning calorimetry (MDSC). Exemplary MDSC curves forenzyme-resistant starch type III ingredients or bulking agents, andisolates thereof, produced in accordance with the present invention areshown in FIGS. 1A, 1B (isolate), 2A, 2B, 3, 4, 5A, 5B (isolate), 5C(isolate), 5D, 5E, and 5F (isolate). Production of the products isdescribed in Examples 1-4 and 6. As exemplified in the Figures, theenzyme-resistant starch type III compositions of the present inventiongenerally melt within a temperature range of about 130° C. to about 160°C. and have an endothermic peak temperature or melting point of at leastabout 140° C. As shown in the Figures, essentially no other peaks occurdown to 50° C. or below, except in a few samples, for which a small peakmay be present, indicating the presence of a small amount ofamylose-lipid complexes.

The RS III is nucleated from a gelatinized starch composition which isat least substantially free of amylopectin crystals and amylose-lipidcomplexes, so as to increase the yield of RS type III. In accordancewith the methods of the present invention, yields of RS type III may beat least about 25% by weight, preferably at least about 30% by weight,most preferably at least about 35% by weight, based upon the weight ofthe original or staring starch ingredient. The yields are determined bythe more stringent Prosky fiber analysis. Consistently high yields maybe obtained on a mass production basis in a batch process, or in asemi-continuous or continuous manner, using relatively few temperaturecycles in a short period of time.

The enzyme-resistant starch type III produced in the present inventionis resistant to enzymes such as α-amylase, β-amylase, amyloglucosidase,and pancreatin and provides a reduced-calorie or low-calorie, highlyfunctional ingredient for baked goods.

The starches used in preparing the enzyme-resistant starch may bederived from any source. Exemplary of starches which may be employed arecorn, potato, sweet potato, wheat, rice, sago, tapioca, waxy maize,sorghum, legume starch, brewer's spent grain, and mixtures thereof.Examples of legume starches which may be employed are pea starches, suchas wrinkled pea or smooth pea starch, faba bean, mung bean, red kidneybean, and lentil bean starch. The amylose content of various cerealstarches, legume starches, and root and tuber starches is disclosed, forexample, in Starch: Properties and Potential, T. Galliard, ed., JohnWiley & Sons, pg. 17 (1987) and Cereal Chem., Vol. 56, No. 5, pg. 477(1979), herein incorporated by reference in their entireties. The starchmay be defatted or chemically modified, for example, converted,derivatized, or crosslinked, and still yield resistant starch. Thestarch may also be partially or completely pregelatinized. However,commercially available pregelatinized starches may be gelatinized attemperatures which melt or destroy crystals of naturally presentresistant starch type III. Accordingly, it is generally preferable touse raw starches as starting starches in the processes of the presentinvention.

Starches which have high contents of amylose or high contents ofamylopectins which have long, straight branch chains are preferred. Thelong, straight branch chains of the amylopectins function as amylose, interms of crystallization, and analyze as amylose by the iodine test. Thestarting starch for use in the present invention preferably has a highcontent of straight chains, to provide a resistant starch havingcrystalline chains of at least about 20 glucose units, preferably atleast about 100 glucose units, derived from amylose and/or fromamylopectin.

Preferred as a starting starch is a starch containing greater than 40%amylose, preferably at least about 50% amylose, most preferably at leastabout 60% by weight amylose, based upon the total weight of amylose andamylopectin. The staring starch also preferably has a low lipid content,for example less than about 0.1% by weight, preferably less than about0.05% by weight so as to avoid the production of undesirableamylose-lipid complexes. Examples of preferred starting starches for usein the present invention are amylomaize starch and wrinkled pea starch,because of their high amylose contents or high apparent amylosecontents. Amylomaize may have an amylose content of about 52% by weightto about 80% by weight and a lipid content of about 0.09% by weight. Theamylose content of wrinkled pea starch may be from about 63% by weightto about 75% by weight. In addition, the lipid content of wrinkled peastarch is only about 0.01% by weight, which is advantageous for avoidingthe formation of amylose-lipid complexes. Commercially available highamylose-content starches which may be used as the starting starch in theprocesses of the present invention are HYLON V, a corn starch containingabout 50% amylose, or HYLON VII, a corn starch containing about 70%amylose, both products of National Starch and Chemical Company,Bridgewater, N.J.

The starting starch may be gelatinized by admixing it with water to forman aqueous slurry at sufficient temperature and pressure to effectgelatinization. Generally, starch gelatinization occurs when: a) waterin a sufficient amount, generally at least about 30% by weight, basedupon the weight of the starch, is added to and mixed with starch and, b)the temperature of the starch is raised to at least about 80° C. (176°F.), preferably 100° C. (212° F.) or more. The gelatinizationtemperature depends upon the amount of water available for interactionwith the starch. The lower the amount of available water, generally, thehigher the gelatinization temperature. Gelatinization may be defined asthe collapse (disruption) of molecular orders within the starch granule,manifested in irreversible changes in properties such as granularswelling, native crystallite melting, loss of birefringence, and starchsolubilization. The temperature of the initial stage of gelatinizationand the temperature range over which it occurs are governed by starchconcentration, method of observation, granule type, and heterogeneitieswithin the granule population under observation. Pasting is thesecond-stage phenomenon following gelatinization in the dissolution ofstarch. It involves increased granular swelling, exudation of molecularcomponents (i.e. amylose, followed by amylopectin) from the granule, andeventually, total disruption of the granules. See Atwell et al., “TheTerminology And Methodology Associated With Basic Starch Phenomena,”Cereal Foods World, Vol. 33, No. 3, pgs. 306-311 (March 1988). Inembodiments of the present invention, the starch granules of thepregelatinized starches may be at least about 90% gelatinized,preferably at least about 95% gelatinized, most preferably completelygelatinized.

In embodiments of the invention, the starting starch and water areadmixed to form an aqueous starch dispersion which is heated togelatinize the starch. If the water content is too low, gelatinizationmay require excessively high temperatures which may destroy or meltcrystals of native or inherent resistant starch type III. Excessivelyhigh water contents during gelatinization can result in prolonged dryingtimes for the resistant-starch composition. The weight ratio of thestarch to water during gelatinization may range from about 0.15:1 toabout 1:1, preferably from about 0.4:1 to about 0.7:1.

The gelatinization of the starting starch ingredient should be conductedat a temperature which is sufficiently high to melt any amylopectincrystals and amylose-liquid complexes, without substantial melting ordestruction of any high-melting enzyme-resistant starch type IIIinherently or naturally present in the starch source. Althoughgelatinization may be effected by any of the methods known in the art,such as batchwise in an upright mixer, it is preferred to use acontinuous process such as passing the aqueous starch mixture through acontinuous mixer. In preferred embodiments, the starting starch andwater may be conveyed into a low-shear continuous mixer/heat exchangeror continuous kneader/heat exchanger such as produced by LIST, Inc.,Acton, Mass. The counterrotating twin screws of the LIST low shear,continuous mixer/kneader may be equipped with hook-like elements forgentle mixing and conveying of the ingredients. The jacketed barrels andthe rotary shafts may be heated by an ethylene glycol heating medium orsteam. In other embodiments of the invention, the starch may begelatinized in a pressure cooker, which helps to prevent the escape ofsteam and thereby maintain a substantially constant starch-to-waterratio.

In embodiments where direct steam injection is used, the steam may besupplied to the LIST mixer or other mixing device at a temperature ofabout 212° F. to about 350° F. Supply pressures may range up to about125 pounds per square inch. The steaming temperatures and pressures canvary depending on the equipment used. As the steam contacts the coldercomposition, the steam condenses and increases the water content of thecomposition.

In other embodiments, the gelatinization may be conducted by forcing thestarch slurry through a jet-cooker. Jet-cookers are well known in theindustry and consist of a cooking chamber in which the starch slurry iscontacted with live steam under elevated temperatures.

Generally, the conditions which may be used for gelatinization aretemperatures from about 110° C. to about 130° C., and pressures fromabout 1.05 kg/cm² to about 21 kg/cm² (about 15 psi to about 30 psi).

After gelatinization, the gelatinized starch is subjected to nucleationand propagation to form and grow crystals of the high-melting-pointresistant starch type III. During nucleation and propagation, the weightratio of the starch to water may be within the same range as duringgelatinization. It is generally not necessary to add or remove waterafter the gelatinization to conduct the nucleation and propagationsteps. However, adjustments in water level may be made to obtain properviscosities for mixing and pumping of the aqueous dispersion of starch.Also, if the water content is too low, mobility of the starch moleculesmay be impeded, which may adversely affect nucleation and propagation.During nucleation and propagation, the starch-to-water weight ratio maybe from about 0.15:1 to about 1:1, preferably from about 0.4:1 to about0.7:1.

After gelatinization, it is critical to maintain the temperature of thegelatinized starch above the melting point of amylopectin starch toprevent the amylopectin from nucleating and propagating. Thus, fornucleation of the resistant-starch type III crystals, the gelatinizedstarch is cooled to a crystal-nucleating temperature which is above themelting point of amylopectin starch. The amylopectin, specificallyamylopectin B crystals, has a melting point substantially below themelting point of enzyme-resistant starch type III. If the gelatinizedstarch is cooled below the melting point of the amylopectin, thensubstantial amounts of crystals of the amylopectin will tend to nucleateand propagate. The nucleation and growth of amylopectin B crystals isbelieved to compete with or impede the nucleation and growth ofhigh-melting amylose crystals, and high-melting linear branches ofamylopectin crystals. By maintaining the crystal-nucleation temperatureabove the melting point of the amylopectin B crystals, the yield andquality of the resistant starch type III crystals is substantiallyincreased.

In addition, the nucleation is preferably conducted at a temperaturewhich is not favorable to nucleation of amylose-lipid complexes. Thus,slow growth, if any, is maintained for the amylose lipid complexes whilepromoting nucleation of the high melting point RS III crystals toprovide numerous growth sites for propagation of the high melting pointRS III crystals of the present invention.

Exemplary enzyme-resistant starch type III nucleating temperatures whichmay be used in the present invention are from about 55° C. to about 100°C., preferably from about 60° C. to about 80° C. The gelatinized starchmay be maintained at the nucleating temperature for a period of timewhich is sufficient to nucleate a substantial amount of crystals of thehigh-melting-point enzyme-resistant starch. Exemplary nucleation timesmay be from about 0.5 hours to about 3 hours. Longer nucleation times,for example up to about 24 hours, may be used. Generally, nucleationtimes of about 1 hour may be employed in embodiments of the presentinvention to obtain high yields in relatively short processing times.

The rate of cooling of the gelatinized starch to the nucleatingtemperature should be as fast as possible and may be at least about 1°C./min on average, preferably at least about 3° C./min on average, mostpreferably at least about 4° C./min on average. By cooling to thenucleation temperature rapidly, the propagation of undesirable crystalforms, such as amylose-lipid complexes, is substantially reduced oreliminated. Also, rapid cooling generally promotes the generation oflarge numbers of small seed crystals, rather than fewer, largercrystals.

After nucleation, the nucleated crystals of enzyme-resistant starch typeIII may be propagated or grown by raising the temperature of thegelatinized starch from the nucleation temperature to acrystal-propagating temperature. Propagation of the resistant starchtype III crystals may be achieved at a temperature above the meltingpoint of any amylose-lipid complexes which may have been formed duringnucleation. Thus, use of a crystal-propagating temperature above themelting point of the amylose-lipid complexes remelts the complexes,thereby making more amylose available for the formation of resistantstarch type III. However, the crystal-propagation temperature ismaintained below the melting point of the desired crystals ofenzyme-resistant starch type III to avoid melting or destroying them.The temperature is preferably raised from the nucleating temperature tothe crystal-propagating temperature at a rapid rate to avoid anysubstantial propagation of undesirable crystals, such as amylose-lipidcomplexes. Exemplary heating rates which may be used are at least about1° C./min on average, preferably at least about 3° C./min on average,most preferably at least about 4° C./min on average. Exemplarycrystal-propagating temperatures for growing crystals of theenzyme-resistant starch type III may range from about 115° C. to about135° C., preferably from about 120° C. to about 130° C. Exemplary timesfor maintaining the temperature at the crystal-propagating temperatureare generally less than about 12 hours, preferably less than about 5hours, most preferably from about 0.5 to about 3 hours.

Temperature cycling may be used in embodiments of the present inventionto increase yields of the high-melting enzyme-resistant starch type III.The increased yield achieves higher calorie reduction, without the needto isolate the high-melting enzyme-resistant starch type III from thegelatinized, amorphous starch or lower-melting starch crystals. Thus,the steps of 1) cooling the gelatinized starch to a nucleatingtemperature, 2) maintaining the gelatinized starch at the nucleatingtemperature, 3) raising the temperature of the gelatinized starch from anucleating temperature to a crystal-propagating temperature, and 4)maintaining the temperature at the crystal-propagating temperature togrow crystals may be performed sequentially in one or more cycles. Forexample, these steps may be performed only once, or sequentiallyrepeated at least once. Preferably, a total of from two to four cyclesof nucleation and propagation are performed to increase yields of thehigh-melting enzyme-resistant starch. In embodiments of the invention,up to about 10 to 12 cycles may be utilized. Generally, increasing thenumber of cycles increases yields of the enzyme-resistant starch typeIII. The nucleating temperatures and times, propagating temperatures andtimes, cooling rates, and heating rates in each cycle may be the same ordifferent, but are preferably at least substantially the same. Forexample, it is preferable to use the same nucleating temperature in eachcycle and the same propagating temperature in each cycle.

After the last step of crystal propagation, the gelatinized starch maybe cooled to a temperature of from about room temperature or about 20°C. to about 50° C. The gelatinized starch may then be dried to obtain abulking agent for producing a flour substitute or baked goods of reducedcalorie content. The cooling is preferably performed rapidly, so as toavoid the propagation of undesirable crystals such as amylose-lipidcomplexes. Exemplary cooling rates may be at least about 1° C./min onaverage, preferably at least about 3° C./min on average, most preferablyat least about 4° C./min on average. In embodiments of the invention,the drying may be performed at room temperature or at elevatedtemperatures. Thus, the gelatinized starch may be cooled from thecrystal-propagating temperature to room temperature or to a dryingtemperature which is above room temperature. Exemplary dryingtemperatures may range from about 20° C. to about 130° C., depending onmode of drying, preferably from about 75° C. to about 85° C., e.g. about80° C., for oven-drying. Known drying methods for the drying of starch,which do not substantially destroy or melt the crystals of resistantstarch type III, may be employed. Exemplary drying methods which may beused include freeze-drying, oven-drying, vacuum-drying, spray-drying,flash-drying, belt drying and drum-drying.

The drying of the high-melting-point resistant starch type IIIcomposition is conducted to achieve a shelf-stable water activity orrelative humidity of less than about 0.7. In embodiments of the presentinvention, the water content of the dried product may approximate thatof commercially available flour. Exemplary moisture contents of thedried, bulking agent or flour substitute or replacer of the presentinvention may range from about 8% by weight to about 14% by weight.

Separation or isolation of the enzyme resistant starch type III from thereadily digestible, amorphous starch or lower melting crystals may beachieved by enzymatic hydrolysis catalyzed by a glycosidase, or amixture of glycosidases or by acid-catalyzed hydrolysis. However, suchseparation or isolation is not preferred and is not needed tosubstantially reduce the calorie content of starch. The temperaturecycling in accordance with the present invention increases yield andcrystal perfection of the resistant starch type III to such a degreethat high calorie reduction is achieved without the need to isolate thehigh melting enzyme resistant starch type III. Additionally, thesubstantial calorie reduction is achieved without substantial loss ofbaking functionality of starch. The dried composition may be useddirectly, or “as is,” as a bulking agent for reduced calorie floursubstitutes or replacers and for reduced calorie baked goods. Thisavoids loss of resistant starch type III crystal solids and increasedcosts associated with removal of the readily digestible starchcomponents.

In embodiments of the invention, seed crystals of resistant starch typeIII may be added in the crystal nucleation step to increase yields ofresistant starch type III in fewer cycles. The RS III seed crystals maybe obtained in at least substantially purified form by isolation from aprevious batch of a starch composition comprising enzyme resistantstarch type III and readily digestible starch components. The seedcrystals may be admixed or dry blended with the starch prior togelatinization of the starch. In other embodiments, the seed crystalsmay be admixed with the gelatinized starch. The seed crystals of thehigh melting enzyme resistant starch type III may be admixed with thegelatinized starch at a temperature preferably above the melting pointof amylopectin crystals and above the melting point of any amylose-lipidcomplexes but below the melting point of RS III crystals to nucleatecrystals of the enzyme resistant starch type III. The seeded mixture maythen be cooled, as described above, to a RS III nucleation temperaturewhich is above the melting point of amylopectin starch. Afternucleation, the temperature may be raised to a crystal propagationtemperature to propagate or grow crystals of resistant starch type IIIas described above. The nucleation and propagation steps may optionallybe repeated, followed by cooling and drying as described above.

In embodiments of the present invention, the gelatinized starch may besubjected to enzymatic debranching prior to or after crystallization ofthe enzyme resistant starch type III to increase its yields. Debranchingprior to crystallization may, for example, result in a RS III yieldincrease of about 1% to 2%. In a preferred embodiment, after subjectingthe gelatinized starch to nucleation and propagation, the resistantstarch type III containing mire is subjected to enzymatic debranching tofurther increase yields of resistant starch type III. For example,debranching after crystallization may increase yields about 5% to about10% by removing amorphous portions of the starch and then subjecting thepartly crystallized starch to additional crystal propagation. It isbelieved that subjecting crystalline resistant starch type III ratherthan noncrystallized starch to debranching facilitates furthercrystallization of the remaining portions of the starch involved inbranch points.

For debranching, the starch solids content may be adjusted to a highersolids level to facilitate subsequent drying of the starch. For example,higher solids levels may be achieved by draining off the water,evaporation, or centrifugation. Exemplary solids levels for debranchingmay be from about 5% by weight to about 40% by weight, preferably fromabout 12% by weight to about 25% by weight solids, based upon the weightof the starch mixture subjected to the debranching enzyme.

After the solids content is fixed, the temperature and pH of the starchdispersion may be readjusted to provide optimum enzyme activity. Theseparameters will vary depending upon the type and source of enzyme used,the enzyme concentration, the substrate concentration, and the presenceor absence of inhibitors.

The enzymatic debranching may be achieved with preferably pullulanase(E.C. 3.2.1.41; pullulan 6-glucanohydrolase), a heat stable enzymeobtained from a species of Bacillus. Pullulanase will catalyze thehydrolysis of the alpha-1,6 linkages in pullulan and amylopectin,provided that there are at least two glucose units in the side chain. Acommercially available pullulanase which may be used is Promozyme 200Lsold by Nordisk, Inc. Other endo-alpha-1,6-glucanohydrolases, such asisoamylase (E.C. 3.2.1.68), or any other endo-enzyme that exhibitsselectivity in cleaving the 1,6-linkages of the starch molecule, leavingthe 1,4-linkages substantially intact, may also be used to debranch thestarch.

The debranching reaction with Bacillus pullulanase may be carried out ina pH range from about 3.0 to about 7.5, preferably from about 4.5 toabout 5.5. Buffers, such as acetates, phosphates, citrates, or the saltsof other weak acids can be added to adjust or maintain the pH at anoptimum level throughout the debranching. During enzymatic debranchingby the Bacillus pullulanase, at a pH of 5.0 for example, the temperatureof the aqueous starch dispersion may range from about 25° C. to about75° C., preferably from about 50° C. to about 65° C. which may be withinthe low end of the nucleation temperature range used in the presentinvention. If shorter treatment times are desired, the optimumtemperature range can be increased to 60°-65° C. or higher, if thedebranching enzyme is thermally stable at the higher temperatures, or ahigher enzyme concentration can be used. As with other parameters of theenzyme reaction, the preferred and optimum temperature ranges will varywith changes in other parameters that affect enzyme activity, such assubstrate concentration and pH, and these can be determined by thepractitioner. However, it is preferable to conduct the debranching at atemperature above the melting point of amylopectin crystals so as toavoid nucleation and propagation of them. Thus, debranching bypullulanase and further nucleation of crystals or crystalline regions ofresistant starch type III may be conducted at a temperature of about 55°C. to about 65° C.

Optimum concentrations of enzyme and substrate are governed by the levelof enzyme activity. The enzyme activity depends upon the enzyme source,the enzyme supplier, and the concentration of the enzyme provided incommercially available batches. In general, pullulanase is effective at1500 PUN (pullulanase units novo/kg starch) using a HYLON V or VIIstarch substrate at 15% solids content. The enzyme may be used insolution, or the enzyme may be immobilized on a solid support.

The enzymatic treatment may be permitted to continue to substantially oressentially completely debranch the starch. Generally, debranching maybe achieved within about 4 to 8 hours. After the desired extent ofstarch debranching has been accomplished, the enzyme may be deactivatedby known deactivation techniques. For example, deactivation or pHadjustment, may be used under conditions which do not substantiallydestroy or melt the crystals of high melting enzyme resistant starchtype III. Bacillus pullulanase, for example, may be rapidly deactivatedat temperatures above about 70° C. (158° F.). The reaction usingpullulanase may therefore be terminated by increasing the temperature ofthe starch dispersion to at least about 75° C. but below the meltingpoint of the high melting resistant starch type III, for about 15minutes. In other embodiments, the enzyme may be deactivated byadjusting the pH of the starch dispersion to below 3.0 and holding atthat pH for about ½ hour. In preferred embodiments, it is preferable tofurther subject the debranched, resistant starch type III composition tocrystal propagation temperatures to grow additional regions or crystalsof resistant starch III. Thus, after debranching, the temperature of thecomposition comprising debranched, enzyme resistant starch type III maybe raised to an enzyme deactivating and crystal propagating temperatureof from about 115° C. to about 135° C., preferably from about 120° C. toabout 130°. Heating rates and times for the propagation, and subsequentcooling rates and times may be the same as described above. Likewise,after debranching and deactivation of the enzyme, the starch may bedehydrated or dried to a shelf-stable moisture content as describedabove to obtain a bulking agent or flour substitute or replacer having areduced calorie content.

If the end-use application requires purification of the starch product,the reaction impurities and by-products of the enzymatic debranchingreaction may be removed by dialysis, filtration, ion exchange processes,centrifugation or any other method known in the art for purifyingstarch. These same purification methods may also be used to isolate orseparate enzyme resistant starch type III crystals, such as seedcrystals, from impurities and by-products of an enzyme-catalyzedhydrolysis or acid-catalyzed hydrolysis.

In preferred embodiments, after completion of the nucleation/propagationtemperature cycling, the enzyme resistant starch type III productobtained may be subjected to heat-treatment. The heat-treatmentsubstantially increases the amount or yield of enzyme resistant starchor total dietary fiber. The heat-treatment also substantially improvesthe baking characteristics or baking functionality of the enzymeresistant starch product in terms of dough cohesiveness, oven spread orgeometry of the baked good, baked good tenderness, and browning.

The higher enzyme resistant starch content or dietary fiber content inthe bulk ingredient may be achieved by the heat-treatment withoutsubstantially adversely affecting the content of enzyme resistant starchtype III which melts at a temperature of at least about 140° C. Theheat-treatment is believed to decrease digestibility of amorphous,non-crystalline starch content of the second stage product. This isevidenced by: 1) a rise in the enzyme resistant starch content or totaldietary fiber content after heat-treatment, but 2) no increase in theenthalpy of the crystalline enzyme-resistant starch type III meltingwithin the temperature range of about 130° C. to about 160° C. afterheat-treatment, and 3) the absence of any additional endothermic peaksin the MDSC curves. It is believed that the heat-treatment in thepresence of water results in densification of the amorphous regions ofthe starch thereby making those regions less accessible to enzymes. Thereduced accessibility increases the amount of enzyme resistant starch ortotal dietary fiber. However, no change in the enthalpy is interpretedto mean that the heat-treatment increases the amount of enzyme resistantstarch which is not crystalline.

As shown in FIG. 6, after gelatinization (stage 1) andnucleation/propagation temperature cycling (stage 2), the enzymeresistant starch type III obtained from stage 2 may be prepared for thestage 3 heat-treatment by drying, grinding, and/or conditioning ormoisture content adjustment. In embodiments of the invention, the dryingmay be conducted to obtain a desired or target moisture content forheat-treating. In other embodiments, the stage 2 product may be dried toa moisture content below a desired moisture content and then moisturemay be added to obtain the desired moisture content. The drying may beconducted in a hot air dryer or other conventional dryer at atemperature which does not melt or destroy the RS type III. Exemplarydrying temperatures may range from about 20° C. to about 130° C.,preferably from about 75° C. to about 85° C., e.g. about 80° C. whenoven-drying. The drying may be conducted to obtain a shelf stablemoisture content for storage prior to subsequent heat-treating or onewhich is within a desired range for grinding to a flowable particulatecomposition. Exemplary moisture contents for storage may range fromabout 0% by weight to about 15% by weight, preferably from about 10% byweight to about 13% by weight. Exemplary moisture contents generallyrange from about 0% by weight to about 30% by weight, preferably fromabout 16% by weight to about 20% by weight for grinding prior to heattreatment.

In preferred embodiments, as shown in FIG. 6, the dried, enzymeresistant starch type III is ground and screened prior to heat-treatmentto enhance yield of enzyme resistant starch. Large chunks of starchgenerally do not respond well to heat-treatment due to non-uniformmoisture content and reduced surface area. The grinding may be performedusing conventional starch grinding equipment such as a Hammer mill. Theextent of grinding may generally be such so as to avoid substantialdestruction of the starch crystals. The screening may be conductedthrough a U.S. mesh no. 20 screen and a U.S. mesh no. 80 screen.Exemplary particle sizes which may be used are less than about 355 μmbut greater than about 45 μm.

As shown in FIG. 6, the ground enzyme resistant starch type III may beconditioned by blending with water to obtain a desired or targetmoisture content for heat-treatment. The conditioning step at leastsubstantially uniformly hydrates the enzyme resistant starch type IIIand it may be performed in a Ribbon blender, continuous mixer, or otherconventional blending or mixing equipment. The moisture content of theenzyme resistant starch during heat treatment may be from about 1% byweight to about 30% by weight, preferably from about 14% by weight toabout 24% by weight, most preferably from about 16% by weight to about20% by weight.

The heat-treatment may be conducted at a temperature of from about 100°C. to about 140° C., preferably from about 125° C. to about 135° C.,most preferably from about 128° C. to about 132° C. Heat-treatment timesmay range from about 5 minutes to about 6 hours, preferably from about30 minutes to about 90 minutes, most preferably from about 50 minutes toabout 70 minutes. The heat-treatment may be conducted in an autoclave,LIST mixer, jacketed continuous mixer, extruder, or other conventionalheating vessels where a substantially constant moisture content may bemaintained.

The heat-treated enzyme resistant starch, as shown in FIG. 6 may beground and screened using conventional flour production equipment toobtain a particle size distribution the same as or compatible with theparticle size distribution of conventional wheat flour. The heat-treatedenzyme resistant starch may be dried prior to or after grinding toobtain a shelf stable moisture content.

In other embodiments of the invention, the heat-treatment may also beused to treat other enzyme resistant starch-containing compositions.Thus, commercially available or known compositions comprising enzymeresistant starch type I, II, or IV may be subjected to theheat-treatment of the present invention to increase the dietary fibercontent or enzyme resistant starch content of the compositions and toimprove their baking characteristics. Exemplary enzyme resistant starchcompositions which may be subjected to the heat-treatment of the presentinvention are Novelose 240 which is an enzyme resistant granular starch,and Novelose 330 which is an enzyme resistant retrograded starch, eachproduced by National Starch and Chemical Co., Bridgewater, N.J., andCrystalean which is a retrograded starch produced by Opta foodIngredients, Inc., Cambridge, Mass.

The non-purified resistant starch type III ingredient or bulking agentobtained by the process of the present invention comprises at leastabout 25% by weight, preferably at least 30% by weight, most preferablyat least 45% by weight of enzyme resistant starch type III as determinedby the stringent Prosky method. The balance of the product comprisesgelatinized, amorphous, or non-crystallized starch. In preferredembodiments, it is at least substantially free, most preferablyessentially or completely free of amylose-lipid complexes and othercrystallized starch products having a melting point below that ofresistant starch III.

In embodiments of the invention, the water holding capacity of theresistant starch type III ingredient is less than 3 grams of water pergram of dry matter, depending upon the yield of resistant starch typeIII and the quality of the crystals. Generally, the fewer the amorphousregions and the greater the crystalline regions, the less is the abilityof the resistant starch ingredient to bind or hold water. The lowerwater holding capacities generally result in a lower viscosity dough anda beneficial effect upon spread and baking characteristics. The waterholding capacity of conventional ungelatinized wheat flour may be about0.6 grams of water per gram of dry flour. In preferred embodiments, thewater holding capacity of the gelatinized, enzyme-resistant bulkingagent or flour substitute or replacer approaches that of conventionalungelatinized wheat flour so as to avoid excessive or too little spreadupon baking. Exemplary water holding capacities for the resistant starchtype III ingredient are less than about 250% by weight, and preferablyrange from about 100% by weight to about 200% by weight (e.g., 1.0 gramwater/gram dry RS III ingredient to 2.0 grams water/gram dry RS IIIingredient).

Enthalpy values for the isolated high-melting enzyme-resistant starchmay range from greater than about 5 J/g, preferably from about 8 J/g toabout 15 J/g depending upon the perfection of the crystals or the numberof amorphous regions or sections in the crystal. Generally, higherenthalpy values indicate, that there are fewer amorphous regions, andthe water holding capacity is lower. The enthalpy of the enzymeresistant bulking agent or flour substitute, at a temperature within therange of about 130° C. to about 160° C., may range from about 0.5 J/g toabout 4 J/g, preferably from about 1 J/g to about 3 J/g, most preferablyabout 2.5 J/g, based upon the weight of the bulking agent or floursubstituent.

The enzyme resistance and low caloric value of the very high meltingenzyme resistant starch type III ingredient or component issubstantially unaltered by baking. The pure, or 100% by weight (100%yield) enzyme resistant starch type III (having a melting point orendothermic peak of at least 140° C.) has a calorific value ofessentially zero, or less than about 0.5 calories/gram even afterbaking. The calorific value for starch which is not resistant starchtype III is about 4 calories/gram. Thus, a resistant starch type IIIingredient or bulking agent with at least a 30% yield of RS type IIIwill exhibit a calorific value of less than about 2.8 calories/gram(0.7×4 cal/g+0.3×0 cal/g=2.8 cal/g).

Even though the resistant starch type III ingredient or bulking agentcontains high amounts of gelatinized starch, it exhibits excellentbaling characteristics in terms of oven spread, edge contour, oilrelease, surface cracking, odor, color or browning, mouthfeel andtexture. It may be used alone or preferably in combination withnon-gelatinized, or substantially ungelatinized flour to obtain doughsfor the production of reduced calorie baked goods such as reducedcalorie cookies and crackers.

In embodiments of the present invention, substantial calorie reductionin baked goods may be achieved by replacing a substantial amount ofconventional, non-gelatinized, flour with the resistant starch type IIIingredient or bulking agent. The present invention provides floursubstitutes comprising substantially ungelatinized flour (non-caloriereduced flour) and calorie reducing amounts of the gelatinized,starch-based bulking agent (reduced calorie flour replacer). The floursubstitute may be combined with cookie and cracker ingredients toprovide doughs which exhibit good machinability on conventional doughforming equipment, and excellent baking characteristics. In embodimentsof the invention, the flour substitute may have a calorie reduction ofat least about 12.5%, preferably at least about 15%, most preferably atleast about 25%, for example about 35% to about 40% by weight, comparedto a conventional non-calorie reduced flour. The flour substitute may beused with other reduced calorie bulking agents or sugar substitutes,such as polydextrose, to obtain doughs for producing reduced caloriebaked goods having a calorie reduction of at least about 25%.

The flour substitutes and doughs of the present invention may compriseat least about 12.5% by weight, preferably at least about 15% by weight,most preferably at least about 25% by weight, for example about 35% byweight to about 40% by weight, of enzyme resistant starch having amelting point of at least about 140° C., said weight percentage beingbased upon the total starch content of the flour substitute or dough,respectively.

The flour component or farinaceous materials which may be combined withthe resistant starch type III ingredient or bulking agent in producingthe flour substitutes and doughs of the present invention may be anycomminuted cereal grain or edible seed or vegetable meal, derivativesthereof and mixtures thereof. Exemplary of the flour component orfarinaceous materials which may be used are wheat flour, corn flour,corn masa flour, oat flour, barley flour, rye flour, rice flour, potatoflour, grain sorghum flour, tapioca flour, ah flour, or starches, suchas corn starch, wheat starch, rice starch, potato starch, tapiocastarch, physically and/or chemically modified flours or starches, suchas pregelatinized starches, and mixtures thereof. The flour may bebleached or unbleached. Wheat flour or mixtures of wheat flour withother grain flours are preferred.

The amount of gelatinized starch-based bulking agent, or the RS IIIingredient, may generally be at least about 25% by weight, for exampleat least about 40% by weight, preferably from about 50% by weight toabout 75% by weight, based upon the total weight of the gelatinized bigagent and the flour component, such as conventional, ungelatinized wheatflour.

The total amount of the flour component and the RS III bulking agentused in the compositions of the present invention may range, forexample, from about 20% by weight to about 80% by weight, preferablyfrom about 45% by weight to about 75% by weight, based upon the weightof the dough. Unless otherwise indicated, all weight percentages arebased upon the total weight of all ingredients forming the doughs orformulations of the present invention, except for inclusions such asflavor chips, nuts, raisins, and the like. Thus, “the weight of thedough” does not include the weight of inclusions.

The flour component may be replaced in whole or in part by conventionalflour substitutes or bulking agents, such as polydextrose,hollocellulose, microcrystalline cellulose, mixtures thereof, and thelike. Corn bran, wheat bran, oat bran, rice bran, mixtures thereof, andthe like may also be substituted in whole or in part for the flourcomponent to enhance color, or to affect texture.

Process-compatible ingredients, which can be used to modify the textureof the products produced in the present invention, include sugars suchas sucrose, fructose, lactose, dextrose, galactose, maltodextrins, cornsyrup solids, hydrogenated starch hydrolysates, protein hydrolysates,glucose syrup, mixtures thereof, and the like. Reducing sugars, such asfructose, maltose, lactose, and dextrose, or mixtures of reducing sugarsmay be used to promote browning. Fructose is the preferred reducingsugar, because of its ready availability and its generally more enhancedbrowning and flavor-development effects. Exemplary sources of fructoseinclude invert syrup, high fructose corn syrup, molasses, brown sugar,maple syrup, mixtures thereof, and the like.

The texturing ingredient, such as sugar, may be admixed with the otheringredients in either solid or crystalline form, such as crystalline orgranulated sucrose, granulated brown sugar, or crystalline fructose, orin liquid form, such as sucrose syrup or high fructose corn syrup. Inembodiments of the invention, humectant sugars, such as high fructosecorn syrup, maltose, sorbose, galactose, corn syrup, glucose syrup,invert syrup, honey, molasses, fructose, lactose, dextrose, and mixturesthereof, may be used to promote chewiness in the baked product.

In addition to the humectant sugars, other humectants, or aqueoussolutions of humectants which are not sugars or possess a low degree ofsweetness relative to sucrose, may also be employed in the dough orbatter. For example, glycerol, sugar alcohols such as mannitol,maltitol, xylitol and sorbitol, and other polyols, may be used ashumectants. Additional examples of humectant polyols (i.e. polyhydricalcohols) include glycols, for example propylene glycol, andhydrogenated glucose syrups. Other humectants include sugar esters,dextrins, hydrogenated starch hydrolysates, and other starch hydrolysisproducts.

In embodiments of the present invention, the total sugar solids content,or the texturizing ingredient content, of the doughs of the presentinvention may range from zero up to about 50% by weight, based upon theweight of the dough.

The sugar solids may be replaced in whole or in part by a conventionalsugar substitute or conventional bulking agent such as polydextrose,hollocellulose, microcrystalline cellulose, mixtures thereof, and thelike. Polydextrose is a preferred sugar substitute or bulking agent formaking the reduced calorie baked goods of the present invention.Exemplary replacement amounts may be at least about 25% by weight, forexample at least about 40% by weight, preferably from about 50% byweight to about 75% by weight, of the original sugar solids content.

In embodiments of the invention, the amount of the conventional sugarsubstitute, conventional bulking agent, or conventional floursubstitute, preferably polydextrose, may be from about 10% by weight toabout 35% by weight, preferably from about 15% by weight to about 25% byweight, based upon the weight of the dough.

The moisture contents of the doughs of the present invention should besufficient to provide the desired consistency to enable proper forming,machining, and cutting of the dough. The total moisture content of thedoughs of the present invention will include any water included as aseparately added ingredient, as well as the moisture provided by flour(which usually contains about 12% to about 14% by weight moisture), themoisture content of the RS III ingredient, and the moisture content ofother dough additives included in the formulation, such as high fructosecorn syrup, invert syrups, or other liquid humectants.

Taking into account all sources of moisture in the dough or batter,including separately added water, the total moisture content of thedoughs or batters of the present invention is generally less than about50% by weight, preferably less than about 35% by weight, based upon theweight of the dough or batter. The cracker doughs of the presentinvention generally have a moisture content of about 27% by weight toabout 33% by weight, based upon the weight of the dough. Cookie doughsof the present invention may have a moisture content of less than about30% by weight, generally from about 10% by weight to about 20% byweight, based upon the weight of the dough.

Oleaginous compositions which may be used to obtain the doughs and bakedgoods of the present invention may include any known shortening or fatblends or compositions useful for baking applications, and they mayinclude conventional food-grade emulsifiers. Vegetable oils, lard,marine oils, and mixtures thereof, which are fractionated, partiallyhydrogenated, and/or interesterified, are exemplary of the shorteningsor fats which may be used in the present invention. Edible reduced- orlow-calorie, partially digestible or non-digestible fats,fat-substitutes, or synthetic fats, such as sucrose polyesters ortriacyl glycerides, which are process-compatible may also be used.Mixtures of hard and soft fats or shortenings and oils may be used toachieve a desired consistency or melting profile in the oleaginouscomposition. Exemplary of the edible triglycerides which can be used toobtain the oleaginous compositions for use in the present inventioninclude naturally occurring triglycerides derived from vegetable sourcessuch as soybean oil, palm kernel oil, palm oil, rapeseed oil, saffloweroil, sesame oil, sunflower seed oil, and mixtures thereof. Marine andanimal oils such as sardine oil, menhaden oil, babassu oil, lard, andtallow may also be used. Synthetic triglycerides, as well as naturaltriglycerides of fatty acids, may also be used to obtain the oleaginouscomposition. The fatty acids may have a chain length of from 8 to 24carbon atoms. Solid or semi-solid shortenings or fats at roomtemperatures of, for example, from about 75° F. to about 95° F. may beused. Preferred oleaginous compositions for use in the present inventioncomprise soybean oil.

Baked goods which may be produced in accordance with the presentinvention include reduced calorie baked goods which are also reducedfat, low fat or no-fat products. As used herein, a reduced-fat foodproduct is a product having its fat content reduced by at least 25% byweight from the standard or conventional product. A low-fit product hasa fat content of less than or equal to three grams of fat per referenceamount or label serving. However, for small reference amounts (that is,reference amounts of 30 grams or less or two tablespoons or less), alow-fat product has a fat content of less than or equal to 3 grams per50 grams of product. A no-fat or zero-fat product has a fat content ofless than 0.5 grams of fat per reference amount and per label serving.For accompaniment crackers, such as a saltine cracker, the referenceamount is 15 grams. For crackers used as snacks and for cookies, thereference amount is 30 grams. Thus, the fat content of a low-fat crackeror cookie would therefore be less than or equal to 3 grams of fat per 50grams or less than or equal to about 6% fat, based upon the total weightof the final product. A no-fat accompaniment cracker would have a fatcontent of less than 0.5 grams per 15 grams or less than about 3.33%,based upon the weight of the final product.

In addition to the foregoing, the doughs of the invention may includeother additives conventionally employed in crackers and cookies. Suchadditives may include, for example, milk by-products, egg or eggby-products, cocoa, vanilla or other flavorings, as well as inclusionssuch as nuts, raisins, coconut, flavored chips such as chocolate chips,butterscotch chips and caramel chips, and the like in conventionalamounts.

A source of protein, which is suitable for inclusion in baked goods, maybe included in the doughs of the present invention to promote Maillardbrowning. The source of protein may include non-fat dry milk solids,dried or powdered eggs, mixtures thereof, and the like. The amount ofthe proteinaceous source may, for example, range up to about 5% byweight, based upon the weight of the dough.

The dough compositions of the present invention may contain up to about5% by weight of a leavening system, based upon the weight of the dough.Exemplary of chemical leavening agents or pH-adjusting agents which maybe used include alkaline materials and acidic materials such as sodiumbicarbonate, ammonium bicarbonate, calcium acid phosphate, sodium acidpyrophosphate, diammonium phosphate, tartaric acid, mixtures thereof,and the like. Yeast may be used alone or in combination with chemicalleavening agents.

The doughs of the present invention may include antimycotics orpreservatives, such as calcium propionate, potassium sorbate, sorbicacid, and the like. Exemplary amounts may range up to about 1% by weightof the dough, to assure microbial shelf-stability.

Emulsifiers may be included in effective, emulsifying amounts in thedoughs of the present invention. Exemplary emulsifiers which may be usedinclude, mono- and di-glycerides, polyoxyethylene sorbitan fatty acidesters, lecithin, stearoyl lactylates, and mixtures thereof. Exemplaryof the polyoxyethylene sorbitan fatty acid esters which may be used arewater-soluble polysorbates such as polyoxyethylene (20) sorbitanmonostearate (polysorbate 60), polyoxyethylene (20) sorbitan monooleate(polysorbate 80), and mixtures thereof. Examples of natural lecithinswhich may be used include those derived from plants such as soybean,rapeseed, sunflower, or corn, and those derived from animal sources suchas egg yolk. Soybean-oil-derived lecithins are preferred. Exemplary ofthe stearoyl lactylates are alkali and alkaline-earth stearoyllactylates such as sodium stearoyl lactylate, calcium stearoyllactylate, and mixtures thereof. Exemplary amounts of the emulsifierwhich may be used range up to about 3% by weight of the dough.

Production of the doughs of the present invention may be performed usingconventional dough mixing techniques and equipment used in theproduction of cookie and cracker doughs. For example, the doughs may besheeted, wire cut, extruded, coextruded, or rotary molded usingconventional equipment. The resistant starch type III ingredient ispreferably preblended with the flour component to obtain a substantiallyhomogeneous mixture for mixing with the other dough ingredients.

While baking times and temperatures will vary for different dough orbatter formulations, oven types, etc., in general, commercial cracker-,cookie-, brownie- and cake-baking times may range from about 2.5 minutesto about 15 minutes, and baking temperatures may range from about 250°F. (121° C.) to about 600° F. (315° C.).

The baked products of the present invention may have a relative vaporpressure (“water activity”) of less than about 0.7, preferably less thanabout 0.6, for preservative free microbial shelf-stability. The watercontent of the cracker products is generally less than about 6% byweight, for example, from about 0.25% by weight to about 4% by weight,based upon the weight of the baked product, exclusive of inclusions.Cookie, brownie and cake products generally have a moisture content ofless than about 20% by weight, for example, from about 2% by weight toabout 9% by weight for cookies, based upon the weight of the bakedproduct, exclusive of inclusions.

The cookie dough or batter compositions of the present invention may beused for the production of reduced calorie bar-type cookies, drop-typecookies such as chocolate chip cookies, oatmeal cookies, sugar cookies,fruit cookies, sandwich cookies, brownies, and the like.

The present invention is further illustrated in the following examples,where all parts, ratios, and percentages are by weight, and alltemperatures are in ° C., unless otherwise stated:

EXAMPLE 1 Preparation of Resistant Starch Ingredient and Analysis

In this example, an enzyme resistant starch type III bulking agent, orresistant starch ingredient, comprising resistant starch type III isprepared and analyzed to determine the yield of resistant starch typeIII and the melting profile of the resistant starch type III:

1A. Preparation of Resistant Starch Ingredient or Bulking Agent

In this example, an enzyme resistant starch type III bulling agent, orresistant starch ingredient, was produced using Hylon VII as a staringstarch. The Hylon VII is a corn starch containing about 70% amylose, andis a product of National Starch and Chemical Company, Bridgewater, N.J.The moisture content of the Hylon VII was about 11% by weight. Hylon VIIin an amount of 150 parts by weight (wet basis) and 450 parts by weightof distilled water were admixed in three Teflon® coated baking pans(batches A, B, and C) to obtain a substantially homogenous starchslurry. The weight ratio of the starch to the water was about 0.33:1.The starch slurry was autoclaved at about 130° C. for about 15 minutesto at least substantially gelatinize the starch.

The gelatinized starch was then subjected to four cycles of crystalnucleation and propagation. In the first cycle, the gelatinized starchslurry was cooled at a cooling rate of about 2° C./min to a nucleationtemperature of about 70° C. The gelatinized starch slurry was thenincubated at about 70° C. for three hours in a humidified enclosure tonucleate crystals of resistant starch III. The humidified enclosure wasoperated at about atmospheric pressure and in the presence of a beakerof water to minimize drying of the surface of the starch gel. The starchslurry was then heated at a rate of about 6° C./min to a crystalpropagating temperature of 130° C. in an autoclave. The temperature of130° C. was held for about 3 hours to grow crystals of enzyme resistantstarch type III.

In the second cycle, the starch slurry was then cooled at a rate ofabout 2° C./min to a crystal nucleation temperature of about 70° C. Thestarch slurry was incubated at that temperature of about 70° C. in thehumidified enclosure overnight (about 18 hours) to nucleate crystals ofresistant starch III. The starch slurry was then heated at a rate ofabout 6° C./min to a crystal propagation temperature of 130° C. in anautoclave and held at that temperature of 130° C. for about 3 hours togrow crystals of enzyme resistant starch type III.

In the third cycle, the gelatinized starch slurry was cooled at acooling rate of about 2° C./min to a nucleation temperature of about 70°C. The gelatinized starch slurry was then incubated at about 70° C. forthree hours in the humidified enclosure to nucleate crystals of enzymeresistant starch III. The starch slurry was then heated at a rate ofabout 6° C./min to a crystal propagating temperature of 130° C. in anautoclave and held at that temperature for about 3 hours to growcrystals of enzyme resistant starch type III.

In the fourth cycle, the starch slurry was then cooled at a rate ofabout 2° C./min to a crystal nucleation temperature of about 70° C. Thestarch slurry was incubated at that temperature of about 70° C. in thehumidified enclosure overnight (about 18 hours) to nucleate crystals ofenzyme resistant starch III. The starch slurry was then heated at a rateof about 6° C./min to a crystal propagation temperature of 130° C. in anautoclave and held at that temperature of 130° C. for about 3 hours togrow crystals of enzyme resistant starch III.

The gelatinized starch gel was then cooled at a rate of about 1° C./minto freeze it. The frozen gel was then freeze dried at a temperature ofabout −30° C. and a pressure of about 10 mTorr to a moisture content ofabout 0 to 2% by weight to obtain a resistant starch ingredient orresistant starch bulking agent in accordance with the present invention.A portion of the sample was then subjected to analysis to determine: a)the yield of the enzyme resistant starch type III, and b) the meltingprofile or thermal characteristics of the enzyme resistant starch typeIII as below in Examples 1B and 1C, respectively:

1B. Isolation of Resistant Starch III and Determination of Yield

The enzyme resistant starch type III was isolated from the resistantstarch type III ingredient or bulling agent of Example 1A above todetermine its yield based upon the original or starting starch (HylonVII). The yield of enzyme resistant starch type III for batches A, B,and C were found to be 34.6%, 35.5%, and 35.8%, respectively. Theaverage yield of resistant starch III was found to be about 35.3%.

The method used for the isolation and determination is adopted andmodified from the Prosky method for Total Dietary Fiber in Foods setforth in AOAC, J. Assoc. Anal. Chem. 68(2) p. 399 (1985) and AOAC,Official Methods of Analysis, J. Assoc. Anal. Chem. 15th ed., pp.1105-1106 (1990). The AOAC method for Total Dietary Fiber in Foodsinvolves: a) treatment with 0.1 ml α-amylase, Sigma Chemical Co.,followed by b) treatment with 5 mg protease, Sigma Chemical Co., thentreatment with 0.3 ml amyloglucosidase, Sigma Chemical Co., d)precipitation of soluble fiber by ethanol, and e) filtering and drying.The adopted method used in the present invention is more stringent,involving higher amounts of enzymes and freeze drying, and results inlower values for the yield of resistant starch. The adopted methodemployed in the present invention is:

Adopted and Modified Prosky Method for Total Dietary Fiber

The pH and temperature of the system is adjusted to maximize enzymeactivity. The buffers and enzymes used in the analysis are:

Buffer Preparation Phosphate Buffer Phosphate Buffer pH = 6 pH = 7.5 2%Phosphoric Acid 0.875 g NaHP0₄ 53.2 ml 0.02M 1.18 ml of 85% PhosphoricNaH₂P0₄ Acid 6.05 g NaH₂P0₄ 297.8 ml 0.01M 50 ml distilled H₂O NaHP0₄dissolve in 700 ml dilute to 1 liter with distilled H₂O distilled H₂Odilute to 1 liter check pH = 7.5 with distilled H₂O check pH = 6Enzymes:

1. Termamyl:

-   -   a. endo-alpha-amylase (Bacillus licheniformis)    -   b. maximum activity at 100° C. (extremely heat stable) and pH+6    -   c. hydrolyzes 1,4-alpha-glucosidic linkages in amylose &        amylopectin    -   d. activity=120 KNU/g (available as 120 liquid)    -   e. produced by Novo Nordisk Bioindustrials, Danbury, Conn.

2. Amyloglucosidase (AMG):

-   -   a. exo-1,4,-a-D-glucosidase (Aspergillus niger)    -   b. maximum activity at 60° C. and pH+4.5    -   c. hydrolyzes 1,4-(more readily) & 1,6-alpha-linkages of starch        glucose units are removed in a stepwise manner from non-reducing        end of substrate    -   d. activity=300 AGU/g    -   e. produced by Novo Nordisk Bioindustrials, Danbury, Conn.

3. Protease:

-   -   a. type XIV bacterial (Streptomyces griseus) Pronase E    -   b. non-specific protease    -   c. activity approx. 4 units/mg solid    -   d. SIGMA Chemical Co.        The Determination of Yield:

In determining yield, the dried starch sample is treated with theα-amylase enzyme (Termamyl), the amyloglucosidase (AMG), and theprotease:

1. Treatment with α-Amylase Enzyme (Termamyl)

Approximately 20 ml of the phosphate buffer having a pH of 6 is added toone gram of dried starch sample. The starch is homogenized to break upany large chunks of starch. Then, 0.4 ml of Termamyl α-amylase is addedper gram of the dried starch sample. The mixture is placed in a 100° C.water bath for a minimum of 30 minutes. The mixture is shakedoccasionally. If pieces of starch gel remain after 30 minutes, the timein the water bath is extended until no more pieces of starch gel arevisible.

2. Treatment with Amyloglucosidase

For treatment with the amyloglucosidase, the pH of the reaction mixtureobtained in step 1 above is adjusted to pH=4.5 using the 2% phosphoricacid. Then, 1.0 ml of the amyloglucosidase enzyme (AMG) per gram of thedried starch sample is added to the starch mixture and stirred. Thestarch mixture is placed in a 60° C. water bath for a minimum of 30minutes and shaked occasionally.

3. Removal of Enzymatic Reaction Products by Centrifuging and Washing

After the enzymatic treatment with the α-amylase (Termamyl) and theamyloglucosidase, the starch mixture is centrifuged and washed two timeswith distilled water:

The enzyme-digested sample is poured into a disposable, 50 ml centrifugetube and centrifuged at 1000 g (3000 rpm) for 10 minutes to obtain apellet (residue) and supernatant liquid. The supernatant is poured off,being careful not to dislodge any particles from the pellet. Then, thepellet is subjected to washing by: a) adding 30 ml of distilled water tothe tube, and breaking the pellet apart with a spatula, and b)centifuging the resulting mixture to obtain a pellet and supernatantliquid and pouring off the supernatant. The pellet washing procedure isthen repeated one more time.

4. Treatment with Protease

After the washing of the enzyme digested sample it is then treated withprotease enzyme to remove the α-amylase (Termamyl) and amyloglucosidaseenzymes:

After pouring off the supernatant in the last wash (above), 20 ml of thephosphate buffer of pH=7.5 are added to the pellet or residue. Then, aprotease solution (a solution containing 16 mg protease (Pronase E) in100 ml phosphate buffer pH=7.5) is added in an amount of 1 ml ofsolution per gram of initial dried starch sample. The resulting mixtureis incubated in a 42° C. water bath for at least 4 hours.

5. Removal of Enzymatic Reaction Products by Centrifuging and Washing

After the enzymatic treatment with the protease, the starch mixture iscentrifuged and washed 3 times with distilled water:

The protease-treated sample is poured into a disposable, 50 mlcentrifuge tube and centrifuged at 1000 g (3000 rpm) for 10 minutes toobtain a pellet (residue) and supernatant liquid. The supernatant ispoured off, being careful not to dislodge any particles from the pellet.Then, the pellet is subjected to washing by: a) adding 30 ml ofdistilled water to the tube, and breaking the pellet apart with aspatula, and b) centrifuging the resulting mixture to obtain a pelletand supernatant liquid and pouring off the supernatant. The pelletwashing procedure is then repeated two more times to obtain an isolatedmaterial or pellet.

6. Freeze Drying of the Isolated Material

Vacuum filtering showed that only a small amount of isolated materialwas found in the supernatant. To save time, the isolated material, orpellet, is freeze dried for at least fifteen hours instead of beingvacuum filtered.

7. Calculation of Yield of Resistant Starch

The yield of the resistant starch is calculated from the weight of thefreeze dried sample:

${{Yield}\mspace{14mu}{of}\mspace{14mu}{Resistant}\mspace{14mu}{Starch}\mspace{14mu}(\%)} = {\frac{\begin{matrix}{{weight}\mspace{14mu}{of}\mspace{14mu}{freeze}\mspace{20mu}{dried}} \\{\mspace{14mu}{{isolated}\mspace{14mu}{material}}}\end{matrix}\mspace{14mu}}{\begin{matrix}{{weight}\mspace{14mu}{of}\mspace{14mu}{sample}} \\\left( {{dry}\mspace{14mu}{basis}} \right)\end{matrix}\mspace{14mu}} \times 100}$1C. Determination of Melting Profile of Resistant Starch Type III byMDSC

The melting profile or thermal characteristics of the resistant starchIII was determined by modulating differential scanning calorimetry(MDSC). In this technique, the material being analyzed is heated at asteady rate with a programmed saw-tooth pattern of heating and coolingimposed upon the steady rate. The fluctuation in temperature in thesaw-tooth pattern is about 1° C. The MDSC technique allows a moreprecise analysis of the equilibrium melting point because it separatesoverlapping thermal events such as irreversible decomposition.

The instruments and method used to characterize the isolate of Example1B which was isolated from the RS Ingredient or bulking agent of Example1A are:

Instrument: TA Instruments Modulated Differential Scanning Calorimeter(MDSC), which includes the TA Instruments DSC 2920 Controller, TAInstruments 2920 Module and the TA Instruments RCS 1061 unit.

Sample Pans: Perkin-Elmer Stainless Steel High Pressure Capsules withgold-plated copper seals.

Sample Preparation: The freeze dried isolate (Example 1B) of the RSIngredient (Example 1A) is ground to a fine powder for analysis. Thesample is weighed in a DSC sample pan, where water (three times theweight of the sample) is placed in the pan to provide a 1:3 solids towater ratio.

Instrument Calibration: The Modulated DSC is calibrated for Baseline,Cell Constant, Temperature and Heat Capacity in known manner:

-   -   Baseline Calibration: Using two empty sample pans the baseline        slope and baseline offset are determined over a temperature        range from 10° C. to 270° C., with a heating rate of 5° C./min.    -   Cell Constant Calibration: Indium is used to determine the cell        constant.    -   Temperature Calibration: Calibrated at two points, indium and        tin.        The DSC Calibration Data Analysis software program is used to        make the proper DSC calibration corrections with the instrument        in the calibration mode.        Heat Capacity is calibrated and the sample is characterized with        the MDSC in the Modulated mode, using the following method:        Equilibrate 30° C.        Data Storage OFF        Isothermal 5 min.        Modulate +/−1.000 C every 60 sec.        Data Storage ON        Ramp 5° C./min to 110° C.        Data Sampling interval 0.2 sec/pt.        Ramp 5.00° C./min to 220° C.        Isothermal 2.00 min.        Data Storage: OFF        Air Cool: ON        Equilibrate at 30° C.        Air Cool: OFF        initial temperature: 30° C.        Heat Capacity Calibration: With sapphire, high density        polyethylene, and polyethylene terephalate, in known manner.        Sample Characterization The Reversing Heat Flow curve is        integrated from 130° C. to 164° C. to measure the enthalpy of        the enzyme resistant starch type III.

Typical results of the MDSC analysis for a sample from one of the threepans are shown in: a) FIG. 1A for the ingredient or bulking agentobtained in Example 1A above, and b) FIG. 1B for the isolated materialor pellet obtained in Example 1B above.

As shown in FIG. 1A, for the bulk ingredient, the onset of meltingoccurs at about 135.3° C., the endothermic peak or the melting point isabout 150.7° C., and the endpoint of melting occurs at about 160° C. Theenthalpy of the enzyme resistant starch type III ingredient or bulkingagent, as shown in FIG. 1A is about 1.89 J/g. Also, as shown in FIG. 1Aessentially no other peaks occur down to 50° C. indicating thesubstantial absence of amylose-lipid complexes as well as thesubstantial absence of other crystalline forms of starch.

As shown in FIG. 1B, for the isolated enzyme resistant starch, the onsetof melting occurs at about 130.7° C., the endothermic peak or themelting point is about 153.6° C., and the endpoint of melting occurs atabout 166.2° C. The enthalpy of the isolated resistant starch type III,as shown in FIG. 1B is about 9.37 J/g. Also, as shown in FIG. 1Bessentially no other peaks occur down to 50° C. indicating thesubstantial absence of amylose-lipid complexes as well as thesubstantial absence of other crystalline forms of starch.

EXAMPLE 2 Preparation of Resistant Starch Ingredient and Analysis

In this example, an enzyme resistant starch type III bulking agent, orresistant starch ingredient, comprising resistant starch type III isprepared and analyzed to determine the yield of resistant starch typeIII and the melting profile of the resistant starch type III bulkingagent:

2A. Preparation of Resistant Starch Ingredient or Bulking Agent

In this example, an enzyme resistant starch type III bulking agent, orresistant starch ingredient, was produced using Hylon VII (about 70% byweight amylose) as a starting starch. The moisture content of the HylonVII was about 11% by weight. Hylon VII in an amount of 125 parts byweight (wet basis) and 375 parts by weight of distilled water wereadmixed in a Teflon® coated baking pan to obtain a substantiallyhomogenous starch slurry. The weight ratio of the starch to the waterwas about 0.33:1. The starch slurry was autoclaved at about 110° C. forabout 15 minutes to at least substantially gelatinize the starch.

The gelatinized starch was then subjected to four cycles of crystalnucleation and propagation. In the first cycle, the gelatinized starchslurry was cooled at a cooling rate of about 2° C./min to a nucleationtemperature of about 58.2° C. The gelatinized starch slurry was thenincubated at about 58.2° C. for three hours in a convection oven tonucleate crystals of resistant starch III. The incubation in theconvection oven was in the presence of a beaker of water to minimizedrying of the surface of the starch gel. The starch slurry was thenheated at a rate of about 6° C./min to a crystal propagating temperatureof 120° C. and autoclaved at that temperature for about 3 hours to growcrystals of resistant starch type III.

In the second cycle, the starch slurry was then cooled at a rate ofabout 2° C./min to a crystal nucleation temperature of about 58.2° C.The starch slurry was incubated at that temperature of about 58.2° C. inthe convection oven overnight (about 18 hours) to nucleate crystals ofresistant starch III. The starch slurry was then heated at a rate ofabout 6° C./min to a crystal propagation temperature of 120° C. andautoclaved at that temperature of 120° C. for about 3 hours to growcrystals of resistant starch III.

In the third cycle, the gelatinized starch slurry was cooled at acooling rate of about 2° C./min to a nucleation temperature of about58.2° C. The gelatinized starch slurry was then incubated at about 58.2°C. in the convection oven overnight (about 18 hours) to nucleatecrystals of resistant starch III. The starch slurry was then heated at arate of about 6° C./min to a crystal propagating temperature of 120° C.and autoclaved at that temperature for about 3 hours to grow crystals ofresistant starch type III.

In the fourth cycle, the starch slurry was then cooled at a rate ofabout 2° C./min to a crystal nucleation temperature of about 58.2° C.The starch slurry was incubated at that temperature of about 58.2° C. inthe convection oven overnight (about 18 hours) to nucleate crystals ofresistant starch III. The starch slurry was then heated at a rate ofabout 6° C./min to a crystal propagation temperature of 120° C. andautoclaved at that temperature of 120° C. for about 3 hours to growcrystals of resistant starch III.

The gelatinized starch gel was then cooled at a rate of about 1° C./minto freeze it. The frozen gel was then freeze dried at a temperature ofabout −30° C. and a pressure of about 10 mTorr to a moisture content ofabout 0 to 2% by weight to obtain a resistant starch ingredient orresistant starch bulking agent in accordance with the present invention.A portion of the sample was then subjected to analysis to determine: a)the yield of the enzyme resistant starch type III, and b) the meltingprofile or thermal characteristics of the enzyme resistant starch typeIII as below in Examples 2B and 2C, respectively:

2B. Isolation of Resistant Starch III and Determination of Yield

The resistant starch type III was isolated from the resistant starchtype III ingredient or bulking agent of Example 2A above to determineits yield based upon the original or starting starch (Hylon VII). Theisolation of the resistant starch III and the determination of yieldwere performed on two samples (A and B) using the method set forth inExample 1B above. The yield of resistant starch III was found to beabout 29.7% for sample A and about 29.5% for sample B, with the averageyield being about 29.6%.

2C. Determination of Melting Profile of Resistant Starch Type inIngredient by MDSC

The melting profile or thermal characteristics of the resistant starchIII bulking agent or RS III ingredient obtained in Example 2A above wasdetermined by modulating differential scanning calorimetry (MDSC). TheMDSC method was the same as described in Example 1C above except thematerial analyzed was the bulking agent rather than the isolatedmaterial.

The results of the MDSC analysis are shown in FIGS. 2A and 2B for twosamples A and B, respectively of the resistant starch bulking agent(resistant starch ingredient) obtained in Example 2A above. As shown inFIG. 2A, for Sample A, the onset of melting occurs at about 132.2° C.,the endothermic peak or the melting point is about 146.7° C., and theendpoint of melting is about 160° C. Also, as shown in FIG. 2Aessentially no other peaks occur down to 50° C. except for one whichindicates the presence of a small amount of amylose-lipid complexes asshown by the relatively small size of the peak. The onset of melting forthe amylose-lipid complexes occurs at about 101.5° C., the endothermicpeak or melting point is at about 106.9° C. and the endpoint of meltingoccurs at about 120° C. The enthalpy of the resistant starch bulkingagent, as shown in FIG. 2A is about 3.048 J/g.

As shown in FIG. 2B, for Sample B, the onset of melting occurs at about131.6° C., the endothermic peak or the melting point is about 147.97°C., and the endpoint of melting is about 162° C. Also, as shown in FIG.2B essentially no other peaks occur down to 50° C. except for one whichindicates the presence of amylose-lipid complexes. The onset of meltingfor the amylose-lipid complexes occurs at about 103.5° C., theendothermic peak or melting point is at about 113.04° C. and theendpoint of melting is about 126° C. The enthalpy of the resistantstarch bulking agent, as shown in FIG. 2B is about 3.340 J/g.

EXAMPLE 3 Preparation of Resistant Starch Ingredient Using Pullulanase

In this example, an enzyme resistant starch type III bulking agent, orresistant starch ingredient, comprising resistant starch type III isprepared and then reacted with pullulanase to increase the yield ofresistant starch type III in the bulking agent. The bulking agent isanalyzed to determine the yield of resistant starch type III before andafter reaction with the pullulanase. The melting profile of theresistant starch type III bulling agent before treatment with thepullulanase is also determined:

3A. Preparation of Resistant Starch Ingredient or Bulking Agent

In this example, an enzyme resistant starch type III bulking agent, orresistant starch ingredient, was produced using Hylon VII (about 70% byweight amylose) as a staring starch. The moisture content of the HylonVII was about 11% by weight. Hylon VII in an amount of 150 parts byweight (wet basis) and 450 parts by weight of distilled water wereadmixed in Teflon® coated baking pans to obtain a substantiallyhomogenous starch slurry. The weight ratio of the starch to the waterwas about 0.33:1. The starch slurry was autoclaved at about 130° C. forabout 15 minutes to at least substantially gelatinize the starch.

The gelatinized starch was then subjected to four cycles of crystalnucleation and propagation followed by a fifth nucleation step. In thefirst cycle, the gelatinized starch slurry was cooled at a cooling rateof about 2° C./min to a nucleation temperature of about 70° C. Thegelatinized starch slurry was then incubated at about 70° C. for threehours in a humidified enclosure to nucleate crystals of resistant starchIII. The humidified enclosure was operated at about atmospheric pressureand in the presence of a beaker of water to minimize drying of thesurface of the starch gel. The starch slurry was then heated at a rateof about 6° C./min to a crystal propagating temperature of 130° C. in anautoclave. The temperature of 130° C. was held for about 3 hours to growcrystals of enzyme resistant starch type III.

In the second cycle, the starch slurry was then cooled at a rate ofabout 2° C./min to a crystal nucleation temperature of about 70° C. Thestarch slurry was incubated at that temperature of about 70° C. in thehumidified enclosure overnight (about 18 hours) to nucleate crystals ofresistant starch III. The starch slurry was then heated at a rate ofabout 6° C./min to a crystal propagation temperature of 130° C. andautoclaved at that temperature of 130° C. for about 3 hours to growcrystals of enzyme resistant starch III.

In the third cycle, the gelatinized starch slurry was cooled at acooling rate of about 2° C./min to a nucleation temperature of about 70°C. The gelatinized starch slurry was then incubated at about 70° C.overnight (about 18 hours) in the humidified enclosure to nucleatecrystals of enzyme resistant starch III. The starch slurry was thenheated at a rate of about 6° C./min to a crystal propagating temperatureof 130° C. in an autoclave and held at that temperature for about 3hours to grow crystals of enzyme resistant starch type III.

In the fourth cycle, the starch slurry was then cooled at a rate ofabout 2° C./min to a crystal nucleation temperature of about 70° C. Thestarch slurry was incubated at that temperature of about 70° C. in thehumidified enclosure 1.5 hours to nucleate crystals of enzyme resistantstarch III. The starch slurry was then heated at a rate of about 6°C./min to a crystal propagation temperature of 130° C. in an autoclaveand held at that temperature of 130° C. for about 3 hours to growcrystals of enzyme resistant starch type III.

In a fifth nucleation step, the gelatinized starch slurry was cooled ata cooling rate of about 2° C./min to a nucleation temperature of about70° C. The gelatinized starch slurry was then incubated at about 70° C.overnight (about 18 hours) in the humidified enclosure to nucleatecrystals of enzyme resistant starch type III.

The gelatinized starch gel was then cooled at a rate of about 1° C./minto freeze it. The frozen gel was then freeze dried at a temperature ofabout −30° C. and a pressure of about 10 mTorr to a moisture content ofabout 0 to 2% by weight to obtain a resistant starch ingredient orresistant starch bulking agent in accordance with the present invention.A portion of the sample was then subjected to analysis to determine themelting profile or thermal characteristics of the enzyme resistantstarch type III as below in Example 3B. Numerous portions of the sampleor bulking agent were also analyzed for the yield of the resistantstarch III and treated with pullulanase as below in Example 3C:

3B. Determination of Melting Profile of Resistant Starch Type IIIIngredient by MDSC

The melting profile or thermal characteristics of the enzyme resistantstarch III bulking agent or RS III ingredient obtained in Example 3Aabove was determined by modulating differential scanning calorimetry(MDSC). The MDSC method was the same as described in Example 1C aboveexcept the material analyzed was the bulking agent rather than theisolated material.

The results of the MDSC analysis are shown in FIG. 3 for the resistantstarch bulking agent (resistant starch ingredient) obtained in Example3A above. As shown in FIG. 3, the onset of melting occurs at about131.8° C., the endothermic peak or the melting point is about 149.9° C.,and the endpoint of melting occurs at about 161.3° C. Also, as shown inFIG. 3 essentially no other peaks occur down to 50° C. which indicatesthe at least substantial absence of amylose-lipid complexes as well asthe at least substantial absence of other forms of crystalline starch.The enthalpy of the resistant starch bulling agent, as shown in FIG. 3is about 2.84 J/g.

3C. Treatment of Bulking Agent with Pullulanase and Determination ofYields

The resistant starch type III ingredient or bulking agent of Example 3Aabove was used to obtain a control and also divided into twelve samples:Samples PA-1, PA-2, PB-1, PB-2, PC-1, PC-2, PD-1, PD-2, PE-1, PE-2, D-1,and D-2. For the control and for each of the twelve samples, theresistant starch type III was isolated from the resistant starch typeIII ingredient or bulking agent to determine its yield based upon theoriginal, or starting starch (Hylon VII). The isolation of the resistantstarch III and the determination of yield for the control and for thetwelve pullulanase-treated samples were performed using the method setforth in Example 1B above. The yield of resistant starch III is reportedfor the control and for each of the twelve pullulanase-treated samplesin Table 1 below.

To show the effect of pullulanase on yield of resistant starch type III,the RS ingredient or RS bulking agent of each of the twelve samples wasenzymatically treated by pullulanase with various conditions inincubation time (1 hour, 2 hours, 3 hours, and 4 hours) and amount ofpullulanase (4.0 μl, 40.0 μl, and 0.4 ml). The same incubationtemperature of 60° C. was used for each of the twelve samples. Thepullulanase enzyme used was Promozyme 200L, produced by Novo NordiskBioindustrials, Danbury, Conn. The Promozyme 200L is a debranchingpullulanase enzyme (Bacillus acidopullulyticus) having a maximumactivity at 60° C. and pH+4.5. The enzyme catalyzes the hydrolysis of1,6-alpha-linkages in pullulan and amylopectin which has been partiallyhydrolyzed by alpha-amylase and is well suited for debranching starchafter liquefaction. The buffer used was a 0.05M citrate buffer of pH5.0. The procedure used to treat each of the twelve samples of theresistant starch III bulking agent or RS III ingredient with thepullulanase was:

1. Weigh about 1 g of each sample of RS III ingredient and add 4 ml ofthe citrate buffer to each sample to obtain a RS III ingredient slurry,except for samples D-1 and D-2 the amount of buffer added is 9 ml.

2. Add the appropriate amount of the Promozyme 200L pullulanase enzymeto each RS III ingredient slurry. The amounts of enzyme used were: a)4.0 μl for samples PA-1, PB-1, PC-1, and PD-1, b) 40 μl for samplesPA-2, PB-2, PC-2, PD-2, D-1 and D-2, and c) 0.4 ml for samples PE-1 andPE-2.

3. Incubate each of the RS III ingredient slurries at 60° C. Theincubation times used were: a) 1 hour for samples PA-1, PA-2, PE-1, andD-1, b) 2 hours for samples PB-1, PB-2, PE-2, and D-2, c) 3 hours forsamples PC-1 and PC-2, and d) 4 hours for samples PD-1 and PD-2.

4. After the enzymatic reaction, incubate each of the samples at 100° C.for 10 minutes to inactivate the enzyme reaction.

5. Wash each of the samples twice with distilled water.

6. Dry each of the samples in an air oven at about 60° C. for about 24hours and measure the recovery of each sample.

7. Isolate the resistant starch III from each pullulanase-treated sampleto determine the yield of the resistant starch III based upon theoriginal or starting starch (Hylon VII). The isolation of the resistantstarch III and the determination of yield were performed on each of thetwelve pullulanase treated samples using the method set forth in Example1B above. The yield of resistant starch III after the pullulanasetreatment is reported in Table 1 below.

Summarized in Table 1 for the pullulanase treatments of the twelvesamples are: a) the enzyme incubation time or reaction time, b) theamount of enzyme used, c) the weight of each sample recovered after thePromozyme enzyme treatment, d) the weight of resistant starch fromrecovered samples after treatment with the Promozyme, e) the yield ofresistant starch, f) the percentage increase in yield obtained by thePromozyme treatment, and g) the total recovery of resistant starch basedon 1 gram of control sample (before enzyme treatment). The latter iscalculated as:

${{Total}\mspace{14mu}{Recovery}\mspace{14mu}{of}\mspace{14mu}{RS}} = {\frac{({Ys})({Ws})}{({Yc})({Wc})} \times 100\%}$

where

-   -   Ys=resistant starch yield of recovered sample with Promozyme        treatment    -   Ws=weight of recovered sample    -   Yc=resistant yield of control and    -   Wc=weight of control (1 gram)

The Table 1 results for the pullulanase treatment are:

TABLE 1 Recovery Of Samples After Treatment With Promozyme PullulanaseAnd Their RS Yield Enzyme Wt. of samples Wt. of RS from Yield increaseIncubation Enzyme recovered after recovered compared Total recovery ofRS Time Amount enzyme samples RS Yield to control compared to controlSample (hours) (μl) treatment (g) (g) (%, db) (%) (%) Control — — —0.3220 33.9 — 100.0 PA-1 1 4 0.9110 0.3485 40.0 18.0 107.5 PA-2 1 400.9358 0.3549 39.5 16.5 109.0 PB-1 2 4 0.9132 0.3513 40.1 18.3 108.0PB-2 2 40 0.8868 0.3509 41.3 21.8 108.0 PC-1 3 4 0.9087 0.3479 39.8 17.4106.7 PC-2 3 40 0.9156 0.3466 39.4 16.2 106.4 PD-1 4 4 0.8587 0.349842.4 24.5 107.4 PD-2 4 40 0.8648 0.3501 42.2 25.1 107.7 PE-1 1 4000.9401 0.3487 38.6 13.9 107.0 PE-2 2 400 0.9458 0.3505 38.6 13.9 107.7D-1 1 40 0.9253 0.3463 39.0 15.0 106.5 D-2 2 40 0.9255 0.3496 39.3 15.9107.3

As indicated in Table 1, the treatment of the resistant starch IIIingredient with the pullulanase increased yield of the resistant starchIII from about 13.9% to about 25.1%. The largest percentage increase inyield was obtained with the longest incubation times of 4 hours forsamples PD-1 and PD-2. The results also indicate that for a givenincubation time and buffer amount, increasing the amount of enzyme from4.0 μl to 40.0 μl or 0.4 ml may tend to decrease yields as indicated bya comparison of the % yield increase for samples PA-1, PA-2, and PE-1;PB-1, PB-2, and PE-2; PC-1 and PC-2; and PD-1 and PD-2.

EXAMPLE 4 Heat Treatment of Enzyme Resistant Starch to Increase Yield

In this example, an enzyme resistant starch type III bulking agent isheat treated to increase the yield of enzyme resistant starch. Two 1gram portions of the resistant starch type III ingredient or bulkingagent produced in Example 1A (batch B) above were subjected to heattreatment to increase the yield of enzyme resistant starch. The heattreatment was conducted by autoclaving each 1 gram sample at 130° C. forone hour.

After the heat treatment, the enzyme resistant starch was isolated fromthe heat-treated, enzyme resistant starch ingredient or bulking agent todetermine its yield based upon the original or staring starch (HylonVII). The isolation of the enzyme resistant starch and the determinationof yield were performed on the two samples using the method set forth inExample 1B above. The yield of enzyme resistant starch was found to beabout 41.5% for one sample and about 41.7% for the other sample, withthe average yield being about 41.6%. Thus, the heat treatment resultedin an unexpectedly superior increase in the average yield of enzymeresistant starch from the average yield of 35.3% obtained by the processof Example 1A.

The melting profile or thermal characteristics of the isolated,heat-treated resistant starch type III samples were determined bymodulating differential scanning calorimetry (MDSC). The MDSC method wasthe same as described in Example 1C above.

The results of the MDSC analysis for one of the isolated heat-treatedsamples is shown in FIG. 4. As shown in FIG. 4, the onset of meltingoccurs at about 130° C., the endothermic peak or the melting point isabout 152.1° C., and the endpoint of melting is about 162.8° C. Also, asshown in FIG. 4 essentially no other peaks occur down to 50° C.indicating the substantial absence of amylose-lipid complexes as well asthe substantial absence of other crystalline forms of starch. Theenthalpy of the isolated heat-treated, enzyme resistant starch bulkingagent, as shown in FIG. 4 is about 7.04 J/g.

Thus, the heat-treatment of the enzyme resistant starch increased theyield of enzyme resistant starch (from 35.3% without heat-treatment, to41.6% with heat-treatment) but the enthalpy decreased (from 9.37 J/gwithout heat-treatment, to 7.04 J/g with heat-treatment). Also, as shownby a comparison of the MDSC curves shown in FIG. 1B and FIG. 4 noadditional crystalline or amylose-lipid peaks resulted from theheat-treatment. Accordingly, it is believed that the heat treatmentincreases the amount of non-crystalline or amorphous enzyme resistantstarch. The increase in enzyme resistance and dietary fiber content isbelieved to result from a densification of the amorphous portion. Thedensification makes it more difficult for the enzyme to attack theamorphous portion of the starch or the non-crystalline dietary fibercreated by the heat-treatment of the starch.

EXAMPLE 5 Effect of Nucleation Temperature Upon Yield

In this example, the effect of nucleation temperature upon yield ofenzyme resistant starch type III was evaluated by subjecting samples ofa gelatinized starting starch to the same nucleation times andpropagation conditions, but different nucleation temperatures, for agiven number of nucleation steps. The different nucleation temperaturesused were: A) 4° C., B) 20° C. (room temperature), C) 40° C., D) 60° C.,E) 80° C., and F) 100° C. For each nucleation temperature (A-F), sampleswere run using 1, 2, and 3 nucleation steps with 0, 1, and 2 propagationsteps, respectively.

Hylon VII, a corn starch containing about 70% amylose, and having amoisture content of about 11% by weight was used as a starting starch toproduce each sample of resistant starch bulking agent. For each sample,Hylon VII in an amount of 1 g (wet basis) and 3 ml of distilled waterwere admixed to obtain a substantially homogenous starch slurry. Theweight ratio of the starch to the water was about 0.33:1. Each starchslurry was autoclaved at about 130° C. for about 15 minutes to at leastsubstantially gelatinize the starch.

The gelatinized starch samples were then subjected to differentnucleation temperatures (A-F) and different numbers of nucleation steps(N) and propagation steps (P). Each propagation step was conducted at120° C. for 3 hours. For each sample, the resistant starch III wasisolated immediately after all of the nucleation steps were completedfor determination of yield of the resistant starch III, based upon theoriginal, or starting starch (Hylon VII). The isolation of the resistantstarch III and the determination of yield were performed on the samplesusing the method set forth in Example 1B above.

A flow chart of the procedure for obtaining the samples fordetermination of yield is:

Cycle N Samples (One Nucleation Step and No Propagation Step)

Thus, to obtain the cycle N samples, the gelatinized starch slurry wascooled at a cooling rate of about 2° C./min to its nucleationtemperature of either 4° C., 20° C. (room temperature), 40° C., 60° C.,80° C., or 100° C. Each of the gelatinized starch slurry samples wasthen incubated at its nucleation temperature for three hours to nucleatecrystals of enzyme resistant starch type III.

Some of the samples were then cooled at a rate of about 1° C./min tofreeze them. The frozen gel was then freeze dried at a temperature ofabout −30° C. and a pressure of about 10 mTorr to a moisture content ofabout 0 to 2% by weight to obtain samples which had been subjected toonly one nucleation step and no propagation steps (Samples N1-A, N1-B,N1-C, N1-D, N1-E, and N1-F). The resistant starch type III wasimmediately isolated from each of the latter samples to determine theyield of resistant starch III based upon the original, or startingstarch (Hylon VII).

Cycle N/P/N Samples (Two Nucleation Steps, One Propagation Step)

To obtain the cycle NPN samples, the remaining samples, were heated at arate of about 6° C./min to a crystal propagating temperature of 120° C.and autoclaved at that temperature for about 3 hours to grow crystals ofresistant starch type III. These samples were then cooled at a rate ofabout 2° C./min to their nucleation temperature of either 4° C., 20° C.(room temperature), 40° C., 60° C., 80° C., or 100° C. Each of thesesamples was then incubated at its nucleation temperature overnight(about 18 hours) to nucleate crystals of enzyme resistant starch III.

Some of the samples were then cooled at a rate of about 1° C./min tofreeze them. The frozen gel was then freeze dried at a temperature ofabout −30° C. and a pressure of about 10 mTorr to a moisture content ofabout 0 to 2% by weight to obtain samples which had been subjected totwo nucleation steps and one propagation step (Samples N2-A, N2-B, N2-C,N2-D, N2-E, and N2-F). The resistant starch type III was immediatelyisolated from each of the latter samples to determine the yield ofenzyme resistant starch III based upon the original, or starting starch(Hylon VII).

Cycle N/P/N/P/N Samples (Three Nucleation Steps and Two PropagationSteps)

To obtain the N/P/N/P/N samples, the remaining samples, were heated at arate of about 6° C./min to a crystal propagating temperature of 120° C.and autoclaved at that temperature for about 3 hours to grow crystals ofresistant starch type III. These samples were then cooled at a rate ofabout 2° C./min to their nucleation temperature of either 4° C., 20° C.(room temperature), 40° C., 60° C., 80° C., or 100° C. Each of thesesamples was then incubated at its nucleation temperature overnight(about 18 hours) to nucleate crystals of resistant starch III.

Some of the samples were then cooled at a rate of about 1° C./min tofreeze them. The frozen gel was then freeze dried at a temperature ofabout −30° C. and a pressure of about 10 mTorr to a moisture content ofabout 0 to 2% by weight to obtain samples which had been subjected tothree two nucleation steps and two propagation steps (Samples N3-A,N3-B, N3-C, N3-D, N3-E, and N3-F). The resistant starch type III wasimmediately isolated from each of the latter samples to determine theyield of enzyme resistant starch III based upon the original, orstarting starch (Hylon VII).

The isolation of the resistant starch III and the determination of yieldwere performed on the samples using the method set forth in Example 1Babove. MDSC was used as in Example 1C above to characterize thecrystallinity of enzyme resistant starch type III that has a meltingpoint or endothermic peak of over 140° C.

The results of the analyses for yields of enzyme resistant starch IIIobtained at the different nucleation temperatures and different numbersof nucleation steps are presented in Table 2:

TABLE 2 Effect of Nucleation Temperature and Cycles on Yield ofResistant Starch III % Change in Yield of Yield Nucleation PropagationResistant Compared to Temperature Nucleation Steps at Starch Yield at20° C. Sample (° C.) Steps 120° C. for 3 hrs (%, dry basis) (R.T.) N1-A 4° C. 1 0 16.6 −0.6 N1-B 20° C. (R.T.) 1 0 16.7 0 N1-C 40° C. 1 0 16.3−2.4 N1-D 60° C. 1 0 18.0 7.8 N1-E 80° C. 1 0 17.4 4.2 N1-F 100° C.  1 017.5 4.8 N2-A  4° C. 2 1 22.3 0.45 N2-B 20° C. (R.T.) 2 1 22.2 0 N2-C40° C. 2 1 22.1 −0.45 N2-D 60° C. 2 1 24.0 8.1 N2-E 80° C. 2 1 24.5 10.4N2-F 100° C.  2 1 22.9 3.1 N3-A  4° C. 3 2 26.8 0.75 N3-B 20° C. (R.T.)3 2 26.6 0 N3-C 40° C. 3 2 26.7 0.38 N3-D 60° C. 3 2 28.7 7.9 N3-E 80°C. 3 2 30.6 15 N3-F 100° C.  3 2 27.6 3.8

In Table 2, the percentage change in yield is calculated relative to theyield at 20° C. (room temperature) for a given number of nucleationsteps. For example, for Sample N1-D (one nucleation step) the percentagechange in yield is calculated relative to the yield for Sample N1-B (onenucleation step at 20° C.): (18.0−16.7)/16.7×100%=7.8%.

As shown in Table 2, for a given number of nucleation steps: nucleationtemperatures of 60° C. to 100° C. resulted in unexpectedly higher yieldsof resistant starch III compared to the yields obtained at nucleationtemperatures of 4° C. to 40° C. For example, for one nucleation step,the yield of resistant starch III was from 4.2% higher to 7.8% higher atnucleation temperatures of 60° C. to 100° C. compared to the yield at20° C. For two nucleation steps, the yield of resistant starch III wasfrom 3.1% higher to 10.4% higher at nucleation temperatures of 60° C. to100° C. compared to the yield at 20° C. For three nucleation steps, theyield of resistant starch III was from 3.8% higher to 15% higher atnucleation temperatures of 60° C. to 100° C. compared to the yield at20° C. For two or more nucleation steps, the highest yields wereobtained at a nucleation temperature of 80° C. and the yield declined ata nucleation temperature of 100° C.

Also, as shown in Table 2, for a given nucleation temperature, the yieldof resistant starch III that has a melting point over 140° C.substantially increases with an increasing number of nucleation andpropagation steps. For example, at a nucleation temperature of 80° C.,the yield of resistant starch III that has a melting point over 140° C.is unexpectedly about 1.75 times higher (30.6/17.4=1.75) after threenucleation steps and two propagation steps (Sample N3-E yield of 30.6%)compared to the yield obtained after one nucleation step and nopropagation step (Sample N1-B yield of 17.4%).

EXAMPLE 6 Effect of Heat Treatment Upon Yield

In this example, the effect of heat treatment upon yield of resistantstarch type III was evaluated by subjecting samples of a resistantstarch III ingredient or bulling agent having different moisturecontents to the same heat treatment temperature and time. The heattreatment temperature was about 130° C. and the heat treatment time wasone hour for each sample. The resistant starch samples having differentmoisture contents were obtained from the same non-heat treated resistantstarch III ingredient or “two-stage product.” The “two-stage product”was produced using a gelatinization stage (stage 1) and anucleation/propagation stage (stage 2), but no heat treatment or thirdstage.

A LIST mixer model #AP80 equipped with three jacketed barrel segmentsand internally heated mixing shafts was used to produce the “two-stageproduct” or non-heat-treated resistant starch ingredient. The startingstarch was Hylon VII, a corn starch containing about 70% amylose and amoisture content of about 11% by weight, produced by National Starch andChemical Company, Bridgewater, N.J. Hylon VII in an amount of about 18parts by weight (wet basis) and about 33.4 parts by weight of water wereadded to the LIST mixer and admixed and heated to obtain a substantiallyhomogenous starch gel. The weight ratio of the starch to the water wasabout 0.54:1. The pressure in the LIST mixer was about 27 psi duringheating up to the gelatinization temperature. The mixture was heated forabout 1 hour to at least substantially gelatinize the starch while thetemperature of all 3 segments and shafts were 130° C.

The gelatinized starch was then subjected to six cycles of crystalnucleation and propagation within the LIST mixer. In the first cycle,the gelatinized starch was cooled to a nucleation temperature of about70° C. within about 15 minutes. The gelatinized starch was thenmaintained at about 70° C. overnight (about 14 hours) to nucleatecrystals of enzyme resistant starch III. The starch was then heated to acrystal propagating temperature of about 130° C. within about 20minutes. The temperature of 130° C. was applied for about 3 hours togrow crystals of enzyme resistant starch type III.

In the second cycle, starch was cooled to a nucleation temperature ofabout 70° C. within about 20 minutes. The gelatinized starch was thenmaintained at about 70° C. for about 3 hours to nucleate crystals ofenzyme resistant starch III. The starch was then heated to a crystalpropagating temperature of about 130° C. within about 20 minutes. Thetemperature of 130° C. was applied for about 2 hours to grow crystals ofenzyme resistant starch type III.

In the third cycle, the starch was cooled to a nucleation temperature ofabout 70° C. within about 20 minutes. The gelatinized starch was thenmaintained at about 70° C. overnight (about 12.5 hours) to nucleatecrystals of enzyme resistant starch III. The starch was then heated to acrystal propagating temperature of about 130° C. within about 30minutes. The temperature of 130° C. was applied for about 1 hour to growcrystals of enzyme resistant starch type III.

In each of the fourth, fifth and sixth cycles, the starch was cooled toa nucleation temperature of about 7° C. within about 20 minutes. Thegelatinized starch was then maintained at about 70° C. for about 1 hourto nucleate crystals of enzyme resistant starch III. The starch was thenheated to a crystal propagating temperature of about 130° C. withinabout 20 minutes. The temperature of 130° C. was applied for about 1hour to grow crystals of enzyme resistant starch type III.

The samples having different moisture contents were obtained by: 1)subjecting the wet, enzyme resistant starch type III ingredient to ovendrying at 80° C. for varied times to obtain samples with the differentmoisture contents in the target range of about 2% by weight to about 20%by weight, and 2) adding water to a sample which had been dried at 80°C. to a moisture content of about 2% to obtain samples having a desiredtarget water content of about 2% by weight to about 20% by weight.

The samples were subjected to grinding and sieved through a No. 20 Meshscreen and through a No. 80 mesh screen to obtain particles having asieve analysis of less than about 355 μm but greater than 45 μm.

The moisture content of a portion of each sample was measured by dryingin an air oven at 130° C. for one hour. The non-heat-treated resistantstarch ingredient used as a control had a moisture content of about 1.6%by weight. The enzyme resistant starch type III content or yield for thecontrol was about 33.4% by weight as determined by the procedure ofExample 1B.

The heat treatment was conducted by autoclaving about 8 grams of eachsample at a temperature of about 130° C. for one hour without coveringthe samples. Each sample was then cooled to room temperature.

The moisture content of a portion of each heat treated sample was thenmeasured by drying in an air oven at 130° C. for one hour and discardedafter the moisture measurement. Another approximately 1 gram portion ofeach sample was treated to isolate the enzyme resistant starch and todetermine the yield of enzyme resistant starch, based upon the original,or starting starch (Hylon VII). The isolation of the enzyme resistantstarch and the determination of yield were performed on the samplesusing the method set forth in Example 1B above.

The melting profile or thermal characteristics of the enzyme resistantstarch samples were determined by modulating differential scanningcalorimetry (MDSC). The MDSC method was the same as described in Example1C above.

The heat treatment temperature and times, moisture contents before andafter heat treatment, the yields of enzyme resistant starch after heattreatment, and the % change in yield resulting from the heat treatmentcompared to the yield obtained without the heat treatment (control) arepresented in Table 3. Also presented in Table 3, and as shown in FIGS.5A through 5F are the onset of melting, the melting point peak, the endpoint of melting, the bulk ingredient enthalpy of the crystalline melt(J/g) and the enzyme resistant starch enthalpy of the crystalline melt(Jig) for several of the samples:

TABLE 3 Effect of Heat Treatment Moisture Content on Yield of ResistantStarch III Yield of % Change Enzyme Moisture Resistant in Yield MeltingBulk Resistant Content Heat Starch III Compared to Onset of Point EndPoint Ingredient Starch before Treatment Moisture After Heat YieldMelting Peak for of Melting Enthalpy of Enthalpy of Heat Time andContent after Treating without for RS RS for RS Crystalline CrystallineTreatment Temperature Heat Treatment (%, dry Heat Isolate IsolateIsolate Melt Melt Sample (weight %) (hrs, ° C.) (weight %) basis)Treatment (° C.) (° C.) (° C.) (J/g) (J/g) Control 1.6 none no heat 33.40   122° C. 151.8° C. 166.1 2.0 9.9 treatment MC-2 1.6 1 hr at 130° C. 8.3 37.9 13.5 — — — — — MC-2-10 10 1 hr at 130° C. 11.6 42.9 28.4 — — —— — MC-2-20 20 1 hr at 130° C. 17.9 44.3 32.6 127.7 151.3 163   — 8.0MC-10 14.8 1 hr at 130° C. 15.0 45.6 36.5 129.6 151.1 164.3 1.4 8.5MC-15 18.4 1 hr at 130° C. 18.3 45.7 36.8 129.6 151.2 165.2 — 8.9 MC-2024 1 hr at 130° C. 23.0 43.0 28.7 — — — — —

In Table 3, the percentage change in yield is calculated relative to the33.4% yield of resistant starch III in the resistant starch ingredientcontrol which has not been heat treated. For example the percentagechange in yield for Sample MC-2 is calculated as:(37.9−33.4/33.4×100% 13.5%.

As shown in Table 3, the heat treatment of the resistant starch IIIingredient or bulking agent unexpectedly increased the yield of theresistant starch III by about 13.5% to about 36.8% compared to the yieldof resistant starch III without heat treating (control). Also as shownin Table 3, the amount of increase in yield depended upon the moisturecontent of the resistant starch III ingredient during heat treatment.The highest increases in yield were obtained when the moisture contentduring heat treatment was about 15% by weight to about 18.3% by weight.

As shown in Table 3 and in FIGS. 5A through 5F, the heat-treatment ofthe enzyme resistant starch increased the yield of enzyme resistantstarch but the enthalpy decreased (from 9.9 J/g for the control withoutheat-treatment). As shown by a comparison of the MDSC curve shown inFIG. 5A with the MDSC curves of FIGS. 5B-5F no additional crystalline oramylose-lipid peaks resulted from the heat-treatment. In fact, the smallamylose-lipid peak (melting point 99.8° C., enthalpy of 0.81 J/g)observed for the control (FIG. 5A) is absent in the MDSC curves shownfor the heat-treated samples (FIGS. 5B through 5F). These resultsindicate that the heat-treatment destroys amylose-lipid complexes orlower melting crystals, while: 1) increasing the amount ofnon-crystalline enzyme resistant starch, and 2) retaining at least asubstantial portion of the crystalline enzyme resistant starch having amelting point greater than about the temperature of heat treating.

EXAMPLE 7 Effect of Nucleation/Propagation Temperatures Upon Yield

In this example, the effect of nucleation temperature and propagationtemperature upon yield of enzyme resistant starch type III was evaluatedby subjecting samples of a gelatinized starting starch to the samenucleation and propagation times, but different nucleation andpropagation temperatures, for a given number of cycles.

Hylon VII, a corn starch containing about 70% amylose, and having amoisture content of about 11% by weight was used as a starting starch toproduce each sample of resistant starch bulking agent. For each sample,Hylon VII in an amount of 5 g (wet basis) and water were admixed toobtain a substantially homogenous 40% by weight starch slurry. Eachstarch slurry was autoclaved at about 130° C. for about 15 minutes to atleast substantially gelatinize the starch.

The gelatinized starch samples were then subjected to differentnucleation and propagation temperatures and steps. Each propagation stepwas conducted for 3 hours. For each sample, the resistant starch III wasisolated immediately after all of the nucleation steps were completedfor determination of yield of the resistant starch III, based upon theoriginal, or starting starch (Hylon VII). The isolation of the resistantstarch III and the determination of yield were performed on the samplesusing the method set forth in Example 1B above.

The procedure for gelatinization, nucleation/propagation temperaturecycling conditions, and analysis was:

Procedure

1. Weigh 5 g of Hylon VII into pressure tube and add water to make 40%starch slurry.

2. Autoclave at either 120° C. or 130° C. for 15 minutes.

3. Nucleate at either 70° C. or 4° C. for 3 hours.

4. Propagate at either 120° C. or 130° C. for 3 hours.

5. Nucleate at either 70° C. or 4° C. overnight (18 hours).

6. Propagate at either 120° C. or 130° C. for 3 hours.

7. Nucleate at either 70° C. or 4° C. overnight (18 hours).

8. Freeze dry the starch gels.

9. Determine RS yield by Prosky method and analyze the bulk RSingredients by MSDC.

The isolation of the resistant starch III and the determination of yieldwere performed on the samples using the method set forth in Example 1Babove. MDSC was used as in Example 1C above to characterize thecrystallinity of enzyme resistant starch type III that has a meltingpoint or endothermic peak of over 140° C.

The results of the analyses for yields of enzyme resistant starch IIIobtained at the different nucleation and propagation temperatures anddifferent numbers of cycles are presented in Table 4. In Table 4, theN/P cycle number is equal to: a) the number of propagation steps, and b)the number of nucleation steps minus one:

TABLE 4 RS Yield and DSC Characteristics Of Freeze Dried Starch Gels(Bulk RS Ingredient) DSC of Bulk RS Ingredient Propagation NucleationN/P RS Peak Temperature Temperature Cycle Yield Enthalpy Temp. (° C.) (°C.) (number) (%, db) (ΔH, J/g) (° C.) 120 4 0 19.6 — — 120 4 1 23.4 — —120 4 2 26.8 1.80 143.97 120 70 0 20.2 — — 120 70 1 24.8 — — 120 70 227.2 1.83 143.45 130 4 0 19.3 — — 130 4 1 24.8 — — 130 4 2 29.4 2.29145.92 130 70 0 19.5 — — 130 70 1 26.7 — — 130 70 2 31.1 2.61 146.77

As shown in Table 4, for a given number of cycles, the highest yields ofenzyme resistant starch are obtained using the higher nucleationtemperature of 70° C. and the higher propagation temperature of 130° C.Also as shown in Table 4, increasing the number of cycles increases theyield of enzyme resistant starch.

Increased amounts or yields of the high melting enzyme resistant starch(melting point peaks in the range of about 143° C. to about 147° C.) andgreater enthalpy at the high melting point peaks are obtained at a 70°C. nucleation temperature compared to a 4° C. nucleation temperature.This effect is enhanced at a propagation temperature of 130° C. comparedto a propagation temperature of 120° C.

Also, both the mass of enzyme resistant starch (as measured by themodified Prosky test of Example 1B) and the MDSC enthalpy of thecrystalline melt at the melting point peaks (about 143° C. to about 147°C.) each increase. This indicates that the enzyme resistant starchproduced through Stage 2 (gelatinization and nucleation/propagationtemperature cycling, but no heat-treatment) is a high melting (meltingpoint peak greater than about 140° C.) crystalline resistant starch typeIII.

EXAMPLE 8 Heat Treatment of Enzyme Resistant Granular Starch

In this example, a commercially available enzyme resistant granularstarch ingredient (an RS type III ingredient) Novelose 240 (produced byNational Starch and Chemical Co.) is heat treated to increase the yieldof enzyme resistant starch. A 1 gram sample of the Novelose 240resistant starch type II ingredient was subjected to heat treatment toincrease the yield of enzyme resistant starch. The heat treatment wasconducted by autoclaving the one gram sample at 130° C. for one hour.

After the heat treatment, the enzyme resistant starch was isolated fromthe heat-treated, enzyme resistant starch ingredient to determine itsyield based upon the original, or starting starch (Novelose 240). Theisolation of the enzyme resistant starch and the determination of yieldwere performed on the heat-treated sample as well as on anon-heat-treated Novelose 240 sample using the method set forth inExample 1B above. The yield of enzyme resistant starch was found to beabout 38% for the heat-treated sample and about 32% for thenon-heat-treated sample. Thus, the heat treatment resulted in anunexpectedly superior increase in the yield of enzyme resistant starch.

The melting profile or thermal characteristics of the isolated,heat-treated resistant starch type II samples were determined bymodulating differential scanning calorimetry (MDSC). The MDSC method wasthe same as described in Example 1C above.

The results of the MDSC analysis for the isolated, non-heat-treatedNovelose 240 sample is shown in FIG. 7. The results of the MDSC analysisfor the isolated, heat-treated Novelose 240 sample is shown in FIG. 8.As shown in FIGS. 7 and 8, neither sample exhibits any endothermicmelting peak in the range of 50° C. to 190° C. and the enthalpy is 0J/g. This indicates that none of the enzyme resistant starch in theNovelose 240 samples, before or after heat-treatment is a high meltingresistant starch type III. Accordingly, it is believed that the heattreatment increases the amount of non-crystalline or amorphous enzymeresistant starch.

EXAMPLE 9 Heat Treatment of Enzyme Resistant Retrograded Starch

In this example, a commercially available enzyme resistant retrogradedstarch ingredient (an RS type III ingredient) Novelose 330 (produced byNational Starch and Chemical Co.) is heat treated to increase the yieldof enzyme resistant starch. The Novelose 330 has a moisture content ofabout 7% by weight, a resistant starch content of about 25% by themethod of Example 1B, and a dietary fiber content by the less stringentAOAC method of about 33%. The Novelose 330 in an amount of about 150grams was admixed with 20.12 ml of water to adjust its moisture contentto about 18% by weight. The heat treatment was conducted by autoclavingthe resulting sample at 130° C. for one hour. The heat-treated samplewas cooled, for analysis.

The enzyme resistant starch was isolated from the heat-treated, enzymeresistant starch ingredient to determine its yield based upon theoriginal, or starting starch (Novelose 330). The isolation of the enzymeresistant starch and the determination of yield were performed on theheat-treated sample using the method set forth in Example 1B above. Thetotal dietary fiber content was also determined using the AOAC method.The yield of enzyme resistant starch was found to be about 42.4% andtotal dietary fiber content by the AOAC method was found to be about56.6% for the heat-treated sample. Thus, the heat treatment resulted inan unexpectedly superior increase in the yield of enzyme resistantstarch and total dietary fiber. Also, MDSC analysis of non-heat-treatedNovelose 330 and heat-treated Novelose 330 performed as in Example 1Cindicates no melting point peak at a temperature greater than about 140°C., as shown in FIGS. 9 and 10. A peak is shown in FIG. 9 at 105.62° C.for the non-heat-treated sample, and a peak is shown in FIG. 10 at116.71° C. for the heat-treated sample.

EXAMPLE 10 Baking Functionality Resistant Starch Ingredients

In this example, the baking functionality of enzyme resistant starchesprepared in accordance with the present invention was compared to thebaking functionality of commercially available, enzyme resistant starchingredients (Novelose 240 and Novelose 330 (produced by National Starchand Chemical Co.) using conventional, non-heat-treated wheat flour as acontrol. The balding functionality was evaluated by measurement ofcookie width or spread. Resistant starch ingredients which resulted in acookie width as close to the width achieved with the wheat flour controlwere considered to have the best baking functionality.

The resistant starch content or yield for each starch ingredient wasmeasured using the method of Example 1B. The total dietary fiber contentfor each starch ingredient was measured used the AOAC method. The MDSCenthalpy (at a temperature of at least about 140° C.) for the bulkingredient was measured as in Example 1C.

The cookie test baling method used to evaluate baking functionality ofthe enzyme resistant starch compositions was the AACC 10-53 Cookie TestBaking Method:

AACC 10-53 Cookie Test Baking Method

The AACC 10-53 Cookie Test Baking Method was designed at Nabisco BiscuitCompany for evaluation of ingredient functionality and predictivecorrelation between sensory and mechanical texture analysis (mechanicaltexture analysis by Instron 3-point bend or puncture test). The test isan improvement over AACC 10-52 Sugar-Snap Cookie Test Baling Method asconfirmed by USDA Soft Wheat Quality Lab (Wooster, Ohio). The AACC 10-53test was adopted as the official method of the American Association ofCereal Chemists after collaborative testing by the Soft Wheat QualityCommittee in 1992. The equipment, cookie dough composition, mixingprocedure, baking procedure, measurement procedure, etc. used in thetest are:

Equipment

Moisture Analyzer, disposable sample pans for determination of flourmoisture.

Digital Thermometer (Omega model 872A) with thermocouple.

C-100 Hobart Mixer with 3-quart mixing bowl and paddle.

National Test Baking Oven.

Aluminum cookie sheet—26 cm width×30 cm length with 2 gauge bars 12 mmwidth×30 cm length×70 mm height.

Cookie cutter (60 mm internal diameter).

Rolling pin with sleeve (sleeve lines run along length of pin).

Spatulas, brown absorbent paper, aluminum foil, plastic beakers.

TA-XT2 Texture Technologies Corporation. **Optional test for doughrheology**—special pan dimensions width 10 cm, length 10.5 cm, height3.2 cm.

Standard Formulation AACC 10-53 Single Batch to Make 4 Test Cookies.

Stage-1 NFDM (nonfat dry milk powder) 2.25 g Salt 2.81 g Soda (sodiumbicarbonate) 2.25 g FGS (sucrose, fine granulation) 94.50 g fat (allvegetable shortening) 90.00 g Stage-2 ABC (ammonium bicarbonate) 1.13 gHFCS (high fructose corn syrup; 42% fructose, 3.38 g 71.5% solids) Water49.50 g Stage-3 Flour (moisture content 13% by weight) 225.00 gGeneral Mixing Procedure

-   -   Stage-1: blend dry ingredients (NFDM, salt, soda, FGS)        -   add to fat        -   mix in Hobart mixer 3 minutes at low speed        -   scrape paddle and sides of bowl after each minute of mixing    -   Stage-2: dissolve ABC in water; **use tap water**        -   add solution of HFCS;        -   add total solution to Stage-1;        -   mix 1 min at low speed, scraping bowl and paddle after each            30 sec.        -   mix 2 min at med speed, scraping bowl and paddle after each            30 sec.    -   Stage-3: add flour to Stage-2;        -   fold into liquid mixture 3 times;        -   mix 2 min at low speed, scraping bowl and paddle after each            30 sec.            Baketime Determination    -   The standard baketime is defined as the time required to produce        a weight loss of 13.85% during baking of the control formulation        at 400° F.    -   To Measure Standard Baketime        -   Bake control formulation at 400° F. for 10, 11, 12, 13            minutes.        -   Plot % weight loss during baking vs. baking time in minutes.        -   Interpolate baketime required to achieve 13.58% weight loss.    -   Baking Specifications        -   Preheat oven to 400° F. (202° C.).        -   Record weight of cold cookie sheet        -   Place cookie sheet in oven for standard baketime; record            weight of hot sheet.        -   Procedure for preparation of 4 dough blanks for cookie test            baking: Portion four 60 g pieces of dough with minimum            deformation and place on cookie sheet. Lay rolling pin            across gauge bars of cookie sheet allowing weight of pin to            compress the dough pieces without additional compressive            force. Pick up rolling pin and place on gauge bars at end of            cookie sheet, and roll ONLY ONCE away from you. Cut cookies            with 60 mm cutter, and carefully lift scrap dough with small            spatula. Lift cutter straight up to avoid horizontal            distortion.        -   Record weight of dough blanks and cookie sheet.        -   Place dough blanks and cookie sheet in the oven in the            direction of sheeting. Bake cookies at 400° F. for            predetermined baketime.        -   Weigh cookie sheet with cookies on it immediately upon            removal from oven. Carefully remove cookies from sheet with            flat spatula and place on flat brown paper in the same            direction in which they were sheeted and baked.            Geometry Measurements (Taken when Cookies are Cooled at            Least 30 Minutes)

-   Width—diameter perpendicular to direction of sheeting. Lay 4 cookies    in a row with rolling-pin-sleeve lines parallel to length of meter    stick. Record measurement in cm. Target is 32.6 cm, with standard    deviation <0.3 cm.

-   Length—parallel to sheeting. Rotate cookies 90° so    rolling-pin-sleeve lines are perpendicular to meter stick. Record    measurement in cm. Target is 33.0 cm, with standard deviation <0.2    cm.

-   Stack Height—stack 4 cookies, and place stack on side between flat    guides. Record measurement in cm. Gently shuffle cookie order and    repeat measurement Target is 3.90 cm, with standard deviation <0.1    cm.

In testing the resistant starch ingredients, a portion (e.g. 50% byweight) of the wheat flour in the standard dough formulation wasreplaced by the enzyme resistant starch ingredient to obtain a blend.For each of the starch compositions, the amount of wheat flourreplacement, the processing, the enzyme resistant starch content oryield, the AOAC total dietary fiber content, the cookie width and theMDSC enthalpy (at a temperature of at least about 140° C.) for the bulkingredient are set forth in Table 5:

TABLE 5 Baking Functionality of RS Ingredients Used To Replace WheatFlour Wheat Flour AOAC MDSC Replacement Total Cookie Enthalpy Starch (%by RS Dietary Cookie Color @>140° C. Sample Ingredient weight)Processing Content Fiber Width L* a* b* Bulk Ingredient Control Wheat 0None 0% 3% 33.0 cm 72.7 6.9 31.8 0 J/g Flour 1 Non-Heat 50% Stage 1 &32% 37% 32.4 cm — — — 1.9 J/g   Treated Stage 2, oven RS III driedIngredient 2 Heat 50% Stage 1 & 47% 63% 35.5 cm 64.6 8.0 32.6 2.0 J/g  Treated Stage 2 & RS III Stage 3 Ingredient Comparative 1 Novelose 50%granular 34% 64% 29.3 cm 82.4 2.1 27.5 0 J/g 240 starch 3 Heat 50%granular 46% 70% 29.2 cm — — — 0 J/g Treated starch + stage 3 Novelose240 Comparative 2 Novelose 50% Retrograded 28% 33% 26.3 cm 80.7 2.4 18.10 J/g 330 starch 4 Heat 50% Retrograded 43% 57% 28.3 cm — — — 0 J/gTreated starch + stage 3 Novelose 330

In Table 5, L*, a*, and b* are standard color measurements of theCommittee on International Illuminescence.

The Stage 3 heat treatments were all conducted at 130° C. for one hourat moisture contents of about 18% by weight.

As shown in Table 5, samples 1 and 2 were the only enzyme resistantstarch ingredients having a high melting point of over 140° C. asevidenced by the MDSC enthalpy of the bulk ingredient. For example, asshown in FIG. 11 an MDSC analysis of the heat-treated enzyme resistantstarch type III of Sample 2, performed as in Example 1C, indicates anenthalpy of 2.02 J/g and an onset of melting at about 140.4° C., anendothermic peak or melting point of 147.86° C., and an end point ofmelting of about 158° C. Also, no additional crystalline oramylose-lipid peaks are shown in FIG. 11 down to a temperature of 50° C.

As shown in Table 5, the heat-treated RS type III ingredient (Sample 2)exhibits both a higher fiber content and unexpectedly superior balkingcharacteristics (cookie width and baked color closest to control)compared to those of: 1) Comparative Example 2, a commerciallyavailable, retrograded resistant starch (Novelose 330), or 2)Comparative Example 1, a processed granular starch (Novelose 240) whichdo not exhibit the high melting point of the RS type III ingredient.

Furthermore, the Stage 3 heat treatment applied to the other resistantstarch of Comparative Examples 1 and 2, provides an improvement orenhancement in both the dietary fiber content and baking functionality(cookie width) as shown by a comparison of the results with Samples 3and 4, respectively. However, the baling functionalities (cookie width)of the heat-treated Novelose ingredients of Samples 3 and 4 are stillinferior to the baking functionalities (cookie width) of theheat-treated resistant starch type III of Sample 2.

1. A method for producing a starch-based composition comprising anenzyme-resistant starch which has a melting point with an endothermicpeak temperature of at least about 140° C. as determined by modulateddifferential scanning calorimetry (MDSC) comprising cooling gelatinizedstarch to a nucleating temperature above the melting point ofamylopectin starch to nucleate crystals of said enzyme-resistant starch,raising the temperature of the gelatinized starch to acrystal-propagating temperature to grow crystals of saidenzyme-resistant starch while melting amylose-lipid complexes which mayhave been formed during said nucleation of said enzyme resistant starch.2. A method for producing a starch-based composition as claimed in claim1 wherein said starch-based composition exhibits essentially no otherendothermic peaks down to 50° C. as determined by modulated differentialscanning calorimetry (MDSC).
 3. A method for producing a starch-basedcomposition as claimed in claim 1 wherein said nucleating temperatureranges from about 55° C. to about 100° C.
 4. A method for producing astarch-based composition as claimed in claim 1 wherein saidcrystal-propagating temperature for growing crystals of theenzyme-resistant starch ranges from about 115° C. to about 135° C.
 5. Amethod for increasing the enzyme-resistant starch content or totaldietary fiber content of an enzyme-resistant starch composition,comprising subjecting said composition to heat treatment at atemperature of from about 100° C. to about 140° C., the moisture contentof said composition during the heat treatment being from about 1% byweight to about 30% by weight.
 6. A method as claimed in claim 5 whereinthe enzyme-resistant starch composition which is subjected to said heattreatment comprises a retrograded, enzyme-resistant starch.
 7. A methodas claimed in claim 5 wherein the enzyme-resistant starch compositionwhich is subjected to said heat treatment comprises an enzyme-resistantstarch type III having a melting point with an endothermic peaktemperature of at least about 140° C. as determined by modulateddifferential scanning calorimetry (MDSC).
 8. A method as claimed inclaim 5 wherein the enzyme-resistant starch composition which issubjected to said heat treatment comprises a granular, enzyme-resistantstarch.
 9. A method as claimed in claim 5 wherein said heat treatment isconducted at a temperature of from about 125° C. to about 135° C.
 10. Amethod as claimed in claim 5 wherein the water-holding capacity of theheat-treated enzyme-resistant starch composition is less than 3 gramswater per gram of said enzyme-resistant starch composition.